Skip to main content
SpringerLink
Log in

Sub-lethal effects induced by a mixture of three non-steroidal anti-inflammatory drugs (NSAIDs) on the freshwater bivalve Dreissena polymorpha

  • Published:
Ecotoxicology Aims and scope Submit manuscript

Abstract

Non-steroidal anti-inflammatory drugs (NSAIDs) are the sixth top-selling drugs worldwide and are commonly found in freshwater ecosystems in the high ng/l to low μg/l range. Recent studies have investigated both the acute and the chronic toxicity of single NSAIDs on different biological models, but these studies have completely neglected the fact that, in the environment, non-target organisms are exposed to mixtures of drugs that have unforeseeable toxicological behavior. This work investigated the sub-lethal effects induced by a mixture of three common NSAIDs, namely, diclofenac, ibuprofen and paracetamol, on the freshwater bivalve, the zebra mussel (Dreissena polymorpha). The mussels were exposed to three different environmental concentrations of the mixture (Low, Mid and High). A multi-biomarker approach was used to highlight cyto-genotoxic effects and the imbalance of the oxidative status of the treated specimens. The Neutral Red Retention Assay (NRRA) was used as a biomarker of cytotoxicity, whereas the activities of catalase, superoxide dismutase, glutathione peroxidase and glutathione S-transferase were measured to assess the role played by the oxidative stress enzymes. In addition, the single cell gel electrophoresis assay, the DNA Diffusion assay and the micronucleus test were used to investigate possible genotoxic effects. According to our NRRA results, each treatment was able to induce a significant cellular stress in bivalves, probably due to the raise of oxidative stress, as indicated by the alteration of enzyme activities measured in treated specimens. Moreover, the mixture induced significant enhancements of DNA fragmentation, which preluded fixed genetic damage, as highlighted by the increase of both apoptotic and micronucleated cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Bigler J, Whitton J, Lampe JW, Fosdick L, Bostick RM, Potter JD (2001) CYP2C9 and UGT1A6 genotypes modulate the protective effect of aspirin on colon adenoma risk. Cancer Res 61:3566–3569

    CAS  Google Scholar 

  • Binelli A, Ricciardi F, Riva C, Provini A (2005) Screening of POP pollution by AChE and EROD activities in zebra mussels from the Italian great lakes. Chemosphere 61:1074–1082. doi:10.1016/j.chemosphere.2005.03.047

    Article  CAS  Google Scholar 

  • Binelli A, Riva C, Cogni D, Provini A (2008) Assessment of the genotoxic potential of benzo(α)pyrene and pp′-dichlorodiphenyldichloroethylene in Zebra mussel (Dreissena polymorpha). Mutat Res 649:135–145. doi:10.1016/j.mrgentox.2007.08.011

    CAS  Google Scholar 

  • Binelli A, Cogni D, Parolini M, Riva C, Provini A (2009a) In vivo experiments for the evaluation of genotoxic and cytotoxic effects of Triclosan in Zebra mussel hemocytes. Aquat Toxicol 91:238–244. doi:10.1016/j.aquatox.2008.11.008

    Article  CAS  Google Scholar 

  • Binelli A, Parolini M, Cogni D, Pedriali A, Provini A (2009b) A multi-biomarker assessment of the impact of the antibacterial trimethoprim on the non-target organism Zebra mussel (Dreissena polymorpha). Comp Biochem Physiol C 150:329–336. doi:10.1016/j.cbpc.2009.05.011

    CAS  Google Scholar 

  • Black MC, Ferrell JR, Horning RC, Martin LK Jr (1996) DNA strand breakage in freshwater mussels (Anodonta grandis) exposed to lead in the laboratory and field. Environ Toxicol Chem 15:802–808. doi:10.1002/etc.5620150528

    CAS  Google Scholar 

  • Bound JB, Voulvoulis N (2006) Predicted and measured concentrations for selected pharmaceuticals in UK rivers: implications for risk assessment. Water Res 40:2885–2892. doi:10.1016/j.watres.2006.05.036

    Article  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  • Buschini A, Carboni P, Martino A, Poli P, Rossi C (2003) Effects of temperature on baseline and genotoxicant-induced DNA damage in haemocytes of Dreissena polymorpha. Mutat Res 537:81–92. doi:10.1016/S1383-5718(03)00050-

    CAS  Google Scholar 

  • Carballa M, Omil F, Lema JM (2008) Comparison of predicted and measured concentrations of selected pharmaceuticals, fragrances and hormones in Spanish sewage. Chemosphere 72:118–1123. doi:10.1016/j.chemosphere.2008.04.03

    Article  Google Scholar 

  • Chaty S, Rodius F, Vasseur P (2004) A comparative study of the expression of CYP1A and CYP4 genes in aquatic invertebrate (freshwater mussel, Unio tumidus) and vertebrate (rainbow trout, Oncorhynchus mykiss). Aquat Toxicol 69:81–93. doi:10.1016/j.aquatox.2004.04.01

    Article  CAS  Google Scholar 

  • Ching EWK, Siu WHL, Lam PKS, Xu L, Zhang Y, Richardson BJ, Wu RSS (2001) DNA adduct formation and DNA strand breaks in green-lipped mussels (Perna viridis) exposed to benzo[a]pyrene: dose- and time-dependent relationships. Mar Pollut Bull 42:603–610. doi:S0025-326X(00)00209-5

    Article  CAS  Google Scholar 

  • Cleuvers M (2003) Aquatic ecotoxicity of pharmaceuticals including the assessment of combination effects. Toxicol Lett 142:185–194. doi:10.1016/S0378-4274(03)00068-7

    Article  CAS  Google Scholar 

  • Cleuvers M (2004) Mixture toxicity of the anti-inflammatory drugs diclofenac, ibuprofen, naproxen and acetylsalicylic acid. Ecotoxicol Environ Saf 59:309–315. doi:10.1016/S0147-6513(03)00141-6

    Article  CAS  Google Scholar 

  • Elia AC, Galarini R, Dorra AJM, Taticchi MI (2007) Heavy metal contamination and antioxidant response of a freshwater bryozoan (Lophopus crystallinus., Pall Phylactolaemata). Ecotoxicol Environ Saf 66:188–194. doi:10.1016/j.ecoenv.2005.12.004

    Article  CAS  Google Scholar 

  • Escher BI, Bramaz N, Eggen RIL, Richter M (2005) In vitro assessment of modes of toxic action of pharmaceuticals in aquatic life. Environ Sci Technol 39:3090–3100. doi:10.1021/es048590e

    Article  CAS  Google Scholar 

  • Fent K, Weston AA, Caminada D (2006) Ecotoxicology of human pharmaceuticals. Aquat Toxicol 76:122–159. doi:10.1016/j.aquatox.2006.02.006

    Article  CAS  Google Scholar 

  • Ferrari B, Paxeus N, Lo Giudice R, Pollio A, Garric J (2003) Ecotoxicological impact of pharmaceuticals found in treated waste waters: study of carbamazepine, clofibric acid and diclofenac. Ecotoxicol Environ Saf 55:359–370. doi:10.1016/S0147-6513(03)00111-8

    Article  CAS  Google Scholar 

  • Flaherty CM, Dodson SI (2005) Effects of pharmaceuticals on Daphnia survival, growth, and reproduction. Chemosphere 61:200–207. doi:10.1016/j.chemosphere.2005.02.016

    Article  CAS  Google Scholar 

  • Gierse JK, Hauser SD, Creely DP, Koboldt C, Rangwala SH, Isakson PC et al (1995) Expression and selective inhibition of the constitutive and inducible forms of human cyclooxygenase. Biochem J 305:479–484

    CAS  Google Scholar 

  • Gomez CF, Constantine L, Moen M, Vaz A, Wang W, Huggett DB (2011) Ibuprofen metabolism in the liver and gill of rainbow trout, Oncorhynchus mykiss. Bull Environ Contam Toxicol 86:247–251. doi:10.1007/s00128-011-0200-8

    Article  CAS  Google Scholar 

  • Grung M, Kallqvist T, Sakshaug S, Skurtveit S, Thomas V (2008) Environmental assessment of Norweigian priority pharmaceuticals based on the EMEA guideline. Ecotoxicol Environ Saf 71:328–340. doi:10.1016/j.ecoenv.2007.10.015

    Article  CAS  Google Scholar 

  • Han GH, Hur HG, Kim SD (2006) Ecotoxicological risk of pharmaceuticals from wastewater treatment plants in Korea: occurrence and toxicity to Daphnia magna. Environ Toxicol Chem 25:265–271. doi:10.1897/05-193R.1

    Article  CAS  Google Scholar 

  • Hayes JD, Flanagan JU, Jowsey IR (2005) Glutathione transferases. Annu Rev Pharmacol Toxicol 45:51–88. doi:10.1146/annurev.pharmtox.45.120403.095857

    Article  CAS  Google Scholar 

  • Heckmann LH, Callaghan A, Hooper HL, Connon R, Hutchinson TH, Maund SJ, Sibly RM (2007) Chronic toxicity of ibuprofen to Daphnia magna: effects on life history traits and population dynamics. Toxicol Lett 172:137–145. doi:10.1016/j.toxlet.2007.06.001

    Article  CAS  Google Scholar 

  • Hong HN, Kim HN, Park KS, Lee SK, Gu MB (2007) Analysis of the effects diclofenac has on Japanese medaka (Oryzias latipes) using real-time PCR. Chemosphere 67:2115–2121. doi:10.1016/j.chemosphere.2006.12.090

    Article  CAS  Google Scholar 

  • Huber C, Bartha B, Harpaintner R, Schröder P (2009) Metabolism of acetaminophen (paracetamol) in plants—two independent pathways result in the formation of a glutathione and a glucose conjugate. Environ Sci Pollut Res 16:206–213. doi:10.1007/s11356-008-0095-z

    Article  CAS  Google Scholar 

  • Jacqz-Aigrain E, Anderson BJ (2006) Pain control: non-steroidal anti-inflammatory agents. Semin Fetal Neonat M 11:251–256. doi:10.1016/j.siny.2006.02.009

    Article  Google Scholar 

  • Kasprzyk-Hordern B, Dinsdale RM, Guwy AJ (2008) The occurrence of pharmaceuticals, personal care products, endocrine disruptors and illicit drugs in surface water in South Wales, UK. Water Res 42(13):3498–3518. doi:10.1016/j.watres.2008.04.026

    Article  CAS  Google Scholar 

  • Khessiba A, Romeo M, Aïssa P (2005) Effects of some environmental parameters on catalase activity measured in the mussel (Mytilus galloprovincialis) exposed to lindane. Environ Pollut 133:275–281. doi:10.1016/j.envpol.2004.05.035

    Article  CAS  Google Scholar 

  • Kirkland DJ, Hayashi M, Jacobson-Kram D, Kasper P, MacGregor JT, Müller L, Uno Y (2007) Summary of major conclusions from the 4th IWGT, San Francisco, 9–10 September, 2005. Mutat Res 627:5–9. doi:10.1016/j.mrgentox.2006.08.009

    CAS  Google Scholar 

  • Kirsch-Volders M, Sofuni T, Aaderma M, Albertini S, Eastmond D, Fenech M, Ishidate M, Lorge E, Norppa H, Surallés J, Von der Hude W, Wakata A (2000) Report from the in vitro micronucleus assay working group. Environ Mol Mutagen 35:167–172. doi:10.1002/(SICI)1098-2280(2000)35:3<167:AID-EM3>3.0.CO;2-G

    Article  CAS  Google Scholar 

  • Langman MJS (1999) Epidemiology of non-steroidal anti-inflammatory drug damage to stomach and duodenum. Ital J Gastroenterol Hepatol 31(S1):2–5

    Google Scholar 

  • Lowe DM, Pipe RK (1994) Contaminant induced lysosomal membrane damage in marine mussel digestive cells-an in vitro study. Aquat Toxicol 30:357–365

    Article  CAS  Google Scholar 

  • Lowe DM, Moore MN, Evans BM (1992) Contaminant impact on interactions of molecular probes with lysosomes in living hepatocytes from dab Limanda limanda. Mar Ecol Prog Ser 91:135–140

    Article  CAS  Google Scholar 

  • Lowe DM, Fossato VU, Depledge MH (1995) Contaminant-induced lysosomal membrane damage in blood cells of mussels Mytilus galloprovincialis from the Venice Lagoon: an in vitro study. Mar Ecol Prog 129:189–196

    Article  Google Scholar 

  • Mamaca E, Bechmann RK, Torgrimsen S, Aas E, Bjørnstad A, Baussant T, Le Floch S (2005) The neutral red lysosomal retention assay and Comet assay on haemolymph cells from mussels (Mytilus edulis) and fish (Symphodus melops) exposed to styrene. Aquat Toxicol 75:191–201. doi:10.1016/j.aquatox.2005.08.001

    Article  CAS  Google Scholar 

  • Maniyke PT, Kharasch ED, Kalhorn TF, Slattery JT (2000) Contribution of CYP2E1 and CYP3A to acetaminophen reactive metabolite formation. Clin Pharmacol Ther 67:275–281. doi:10.1067/mcp.2000.104736-S0009-9236(00)56394-2

    Article  Google Scholar 

  • McCahon CP, Pascoe D (1988) Use of Gammarus pulex (L.) in safety evaluation tests: culture and selection of a sensitive life stage. Ecotoxicol Environ Saf 15:245–252

    Article  CAS  Google Scholar 

  • Mehinto A, Hill E, Tyler C (2010) Uptake and biological effects of environmentally relevant concentrations of the nonsteroidal anti-inflammatory pharmaceutical diclofenac in rainbow trout (Oncorhynchus mykiss). Environ Sci Technol 44:2176–2182. doi:10.1021/es903702m

    Article  CAS  Google Scholar 

  • Misra R, Pandey H, Chandra M, Agarwal PK, Pandeya SN (1990) Effects of commonly used non steroidal anti-inflammatory drugs on gastric mucosa. A clinical, endoscopic and histopathological study. J Assoc Phys India 38(9):636–638

    CAS  Google Scholar 

  • Mitchelmore CL, Chipman JK (1998) Detection of DNA strand breaks in brown trout (Salmo trutta) hepatocytes and blood cells using the single cell gel electrophoresis (comet) assay. Aquat Toxicol 41:161–182. doi:10.1016/S0166-445X(97)00064-7

    Article  CAS  Google Scholar 

  • Osman AM, van Noort PCM (2007) Comparison of key enzymes in the zebra mussel, Dreissena polymorpha, the earthworm Allolobophora chlorotica and Chironomus riparius larvae. Ecotoxicol Environ Saf 67:212–217. doi:10.1016/j.ecoenv.2006.05.003

    Article  CAS  Google Scholar 

  • Parolini M, Binelli A, Cogni D, Provini A (2010) Multi-biomarker approach for the evaluation of the cyto-genotoxicity of paracetamol on the zebra mussel (Dreissena polymorpha). Chemosphere 79:489–498. doi:10.1016/j.chemosphere.2010.02.053

    Article  CAS  Google Scholar 

  • Parolini M, Binelli A, Provini A (2011a) Assessment of the potential cyto–genotoxicity of the nonsteroidal anti-inflammatory drug (NSAID) diclofenac on the zebra mussel (Dreissena polymorpha). Water Air Soil Pollut 217:589–601. doi:10.1007/s11270-010-0612-9

    Article  CAS  Google Scholar 

  • Parolini M, Binelli A, Provini A (2011b) Chronic effects induced by ibuprofen on the freshwater bivalve Dreissena polymorpha. Ecotoxicol Environ Saf 74(6):1586–1594. doi:10.1016/j.ecoenv.2011.04.025

    Article  CAS  Google Scholar 

  • Pascoe D, Karntanut W, Muller CT (2003) Do pharmaceuticals affect freshwater invertebrates? A study with the cnidarian Hydra vulgaris. Chemosphere 51:521–528. doi:10.1016/S0045-6535(02)00860-3

    Article  CAS  Google Scholar 

  • Pavlica M, Klobucar GIV, Vetma N, Erben R, Papes D (2000) Detection of micronuclei in haemocytes of zebra mussel and ramshorn snail exposed to pentachlorophenol. Mutat Res 465:145–150. doi:10.1016/S1383-5718(99)00222-3

    CAS  Google Scholar 

  • Pöch G (1993) Combined effects of drugs and toxic agents. Modern evaluation in theory and practice. Springer, Berlin

    Book  Google Scholar 

  • Regoli F, Nigro M, Orlando E (1998) Lysosomal and antioxidant responses to metals in the Antarctic scallop Adamussium colbecki. Aquat Toxicol 40:375–392. doi:10.1016/S0166-445X(97)00059-3

    Article  Google Scholar 

  • Regoli F, Pellegrini D, Winston GW, Gorbi S, Giuliani S, Virno–Lamberti C, Bompadre S (2002) Application of biomarkers for assessing the biological impact of dredged materials in the Mediterranean: the relationship between antioxidant responses and susceptibility to oxidative stress in the red mullet (Mullus barbatus). Mar Pollut Bull 44:912–922. doi:10.1016/S0025-326X(02)00120-0

    Article  CAS  Google Scholar 

  • Regoli F, Winston GW, Gorbi S, Frenzilli G, Nigro M, Corsi I, Focardi S (2003) Integrating enzymatic responses to organic chemical exposure with total oxyradical absorbing capacity and DNA damage in the European eel Anguilla anguilla. Environ Toxicol Chem 22:2120–2129. doi:10.1897/02-378

    Article  CAS  Google Scholar 

  • Riva C, Binelli A, Cogni D, Provini A (2007) Evaluation of DNA damages induced by BDE-209 in haemocytes of Dreissena polymorpha using SCGE assay and MN test. Environ Mol Mutagen 48:735–743. doi:10.1002/em.20353

    Article  CAS  Google Scholar 

  • Santos LHMLM, Araujo AN, Fachini A, Pena A, Deleure-Matos C, Montenegro MCBSM (2010) Ecotoxicological aspects related to the presence of pharmaceuticals in the aquatic environment. J Hazard Mat 175(1–3a):45–95. doi:10.1016/j.jhazmat.2009.10.100

    Article  CAS  Google Scholar 

  • Shen S, Hargus SJ, Martin BM, Pohl LR (1997) Cytochrome P4502C11 is a target of diclofenac covalent binding in rats. Chem Res Toxicol 10:420–423. doi:10.1021/tx960167z

    Article  CAS  Google Scholar 

  • Singh NP (2000) A simple method for accurate estimation of apoptotic cells. Exp Cell Res 256:328–337. doi:10.1006/excr.2000.4810

    Article  CAS  Google Scholar 

  • Singh NP, McCoy MT, Tice RR, Schneider E (1988) A simple technique for quantification of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191

    Article  CAS  Google Scholar 

  • Siu WHL, Hung CLH, Wong HL, Richardson BJ, Lam PKS (2003) Exposure, time-dependent DNA strand breakage in hepatopancreas of green-lipped mussels (Perna viridis) exposed to Aroclor, 1254 and mixtures of B[a]P and Aroclor 1254. Mar Pollut Bull 46:1285–1293. doi:10.1016/S0025-326X(03)00234-0

    Article  CAS  Google Scholar 

  • Siu WHL, Cao J, Jack RW, Wu RSS, Richardson BJ, Xu L, Lam PKS (2004) Application of the comet and micronucleus assays to the detection of B[a]P genotoxicity in haemocytes of the green-lipped mussel (Perna viridis). Aquat Toxicol 66:381–392. doi:10.1016/j.aquatox.2003.10.006

    Article  CAS  Google Scholar 

  • Stuer-Lauridsen F, Birkved M, Hansen LP, Holten Lutzhoft HC, Halling-Sorensen B (2000) Environmental risk assessment of human pharmaceuticals in Denmark after normal therapeutic use. Chemosphere 40(7):783–793. doi:10.1016/S0045-6535(99)00453-1

    Article  CAS  Google Scholar 

  • Todd PA, Sorkin EM (1988) Diclofenac sodium, a reappraisal of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy. Drugs 35:244–285

    Article  CAS  Google Scholar 

  • Valavanidis A, Vlahogianni T, Dassenakis M, Scoullos M (2006) Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants. Ecotoxicol Environ Saf 64:178–189. doi:10.1016/j.ecoenv.2005.03.013

    Article  CAS  Google Scholar 

  • Van Goethem F, Lison D, Kirsh-Volders M (1997) Comparative evaluation of the in vitro micronucleus test and the alkaline single cell gel electrophoresis assay for the detection of DNA damaging agents: genotoxic effects of cobalt powder, tungsten carbide and cobalt–tungsten carbide. Mutat Res 392:31–43

    Google Scholar 

  • Winston GW, Di Giulio RT (1991) Prooxidant and antioxidant mechanism in aquatic organism. Aquat Toxicol 19:137–161. doi:10.1016/0166-445X(91)90033-6

    Article  CAS  Google Scholar 

  • Winston GW, Moore MN, Kirchin MA, Soverchia C (1996) Production of reactive oxygen species (ROS) by hemocytes from the marine mussel, Mytilus edulis. Comp Biochem Physiol C 113:221–229. doi:10.1016/0742-8413(95)02091-8

    Article  CAS  Google Scholar 

  • EMEA (European Agency for the Evaluation of Medicinal Products), n. Note for guidance on environmental risk assessment of medical products for human use

  • Zanger UM, Turpeinen M, Klei K, Schwab M (2008) Functional pharmacogenetics/genomics of human cytochromes P450 involved in drug biotransformation. Anal Bioanal Chem 392:1093–1108. doi:10.1007/s00216-008-2291-6

    Article  CAS  Google Scholar 

  • Zuccato E, Castiglioni S, Bagnati R, Chiabrando C, Grassi P, Fanelli R (2008) Illicit drugs, a novel group of environmental contaminants. Water Res 42:961–968. doi:10.1016/j.watres.2007.09.010

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biology, University of Milan, Via Celoria 26, 20133, Milan, Italy

    Marco Parolini & Andrea Binelli

Authors
  1. Marco Parolini
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrea Binelli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Marco Parolini.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Parolini, M., Binelli, A. Sub-lethal effects induced by a mixture of three non-steroidal anti-inflammatory drugs (NSAIDs) on the freshwater bivalve Dreissena polymorpha . Ecotoxicology 21, 379–392 (2012). https://doi.org/10.1007/s10646-011-0799-6

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10646-011-0799-6

Keywords

Search

Navigation