Ibuprofen and diclofenac in the marine environment - a critical review of their occurrence and potential risk for invertebrate species

Abstract

The presence of the two main non-steroidal anti-inflammatory drugs (NSAIDs), diclofenac (DCF) and ibuprofen (IBU), in marine and estuarine systems has been reported. Although the available information about the toxicity of these compounds in aquatic organisms has increased in recent years, the database about marine organisms, specifically invertebrates, is limited. Consequently, the assessment of potential risks posed by these compounds to aquatic species should be improved, given their relevance for aquatic life and the trophic chain. To fill this gap, we analyze the potential risk of IBU and DCF in marine invertebrates. To assess the risk, the database was built with available information from the scientific literature about the occurrence, bioaccumulation, and toxicity of both compounds in the estuaries and marine environments. Risk assessment of both compounds in these environments is scarce and based essentially on their acute toxicity. Data compiled in this review, including environmental concentrations and toxicity thresholds, were used to calculate risk quotients and classify the risk for invertebrate communities in coastal areas with different contamination levels. The results indicated a higher risk for DCF than IBU. Additionally, the simultaneous presence of the two NSAIDs in the aquatic environment increases the risk for exposed organisms. The use of classical methods (e.g., biochemical markers, gene expression), new approaches (e.g., adverse outcome pathway, AOP), and omic techniques has contributed to understanding the underlying toxicity mechanisms and improving the risk assessment. However, in a global change scenario involving multiple drivers and pressures such as ocean acidification, heat waves, climate change, etc., the risk assessment for these emerging pollutants needs improvement. This can be achieved by increasing our knowledge about the metabolic pathway and biotransformation routes of these compounds in marine species and understanding how these changes can affect the bioaccumulation, toxicity, fate, and behavior of both NSAIDs.

Keywords

Non-steroidal anti-inflammatory drugs / saltwater ecosystems / invertebrates / bioaccumulation / adverse effects / risk assessment

References

[1] Tornero V. Potential chemical contaminants in the marine environment: An overview of main contaminant lists. JRC Technical Reports. Available from: https://mcc.jrc.ec.europa.eu/documents/201801085655.pdf. [Last accessed on 2 Aug 2023]

[2] Vlachogianni T.Pharmaceuticals and personal care products as contaminants in the aquatic environment. A category of organic wastewater pollutants with special characteristics.Pharmakeftiki2013;25:16-23Available from: https://www.researchgate.net/publication/236229893.[Last accessed on 2 Aug 2023]

[3] Liu JL.Pharmaceuticals and personal care products (PPCPs): a review on environmental contamination in China.Environ Int2013;59:208-24

[4] Ali AM,Alarif W,Al-Lihaibi SS.Occurrence of pharmaceuticals and personal care products in effluent-dominated Saudi Arabian coastal waters of the Red Sea.Chemosphere2017;175:505-13

[5] Adeleye AS,Zhao Y.Abundance, fate, and effects of pharmaceuticals and personal care products in aquatic environments.J Hazard Mater2022;424:127284

[6] Daughton CG.Pharmaceuticals and personal care products in the environment: agents of subtle change?.Environ Health Perspect1999;107 Suppl 6:907-38 PMCID:PMC1566206

[7] Gros M,Barceló D.Wastewater treatment plants as a pathway for aquatic contamination by pharmaceuticals in the ebro river basin (northeast Spain).Environ Toxicol Chem2007;26:1553-62

[8] Petrović M,Barceló D.Analysis and removal of emerging contaminants in wastewater and drinking water.TrAC Trends in Analytical Chemistry2003;22:685-96

[9] Osorio V,Aceña J,Barceló D.Concentration and risk of pharmaceuticals in freshwater systems are related to the population density and the livestock units in Iberian Rivers.Sci Total Environ2016;540:267-77

[10] Monteiro SC.Occurrence and fate of human pharmaceuticals in the environment.Rev Environ Contam Toxicol2010;202:53-154

[11] Mezzelani M,Regoli F.Pharmaceuticals in the aquatic environments: evidence of emerged threat and future challenges for marine organisms.Mar Environ Res2018;140:41-60

[12] Fonseca TG,Rocha T,Aureliano M.Ecotoxicological assessment of the anticancer drug cisplatin in the polychaete Nereis diversicolor.Sci Total Environ2017;575:162-72

[13] Jelic A,Ginebreda A.Occurrence, partition and removal of pharmaceuticals in sewage water and sludge during wastewater treatment.Water Res2011;45:1165-76

[14] Santos LHMLM,Fachini A,Delerue-Matos C.Ecotoxicological aspects related to the presence of pharmaceuticals in the aquatic environment.J Hazardous Materials2010;175:45-95

[15] Świacka K,Maculewicz J,Smolarz K.Toxic effects of NSAIDs in non-target species: a review from the perspective of the aquatic environment.Environ Pollut2020;273:115891

[16] Zhao JL,Liu YS.Occurrence and a screening-level risk assessment of human pharmaceuticals in the Pearl River system, South China.Environ Toxicol Chem2010;29:1377-84

[17] Thomas KV.The occurrence of selected human pharmaceutical compounds in UK estuaries.Mar Pollut Bull2004;49:436-44

[18] Beretta M,Tavares TM,Pletsch AL.Occurrence of pharmaceutical and personal care products (PPCPs) in marine sediments in the Todos os Santos Bay and the north coast of Salvador, Bahia, Brazil.J Soils Sediments2014;14:1278-86

[19] Hernández-Tenorio R,Guzmán-Mar JL,Hernández-Ramírez A.Review of occurrence of pharmaceuticals worldwide for estimating concentration ranges in aquatic environments at the end of the last decade.J Hazard Mater2022;8:100172

[20] Commission Implementing Decision (EU) 2015/495 of 20 March 2015 establishing a watch list of substances for Union-wide monitoring in the field of water policy pursuant to Directive 2008/105/EC of the European Parliament and of the Council (notified under document C(2015) 1756) Text with EEA relevance. Available from: https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=uriserv%3AOJ.L_.2015.078.01.0040.01.ENG. [Last accessed on 2 Aug 2023]

[21] Commission Implementing Decision (EU) 2018/840 of 5 June 2018 establishing a watch list of substances for Union-wide monitoring in the field of water policy pursuant to Directive 2008/105/EC of the European Parliament and of the Council and repealing Commission Implementing Decision (EU) 2015/495 (notified under document C(2018) 3362). Available from: https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32018D0840. [Last accessed on 2 Aug 2023]

[22] Batucan NSP,Northcott GL.Medicating the environment? A critical review on the risks of carbamazepine, diclofenac and ibuprofen to aquatic organisms.Environ Adv2022;7:100164

[23] Trombini C,Hampel M.Ibuprofen and diclofenac: effects on freshwater and marine aquatic organisms - Are they at risk? In: Gómez-Oliván LM, editor. Non-Steroidal Anti-Inflammatory Drugs in Water. The Handbook of Environmental Chemistry; 2020. p. 161-89.

[24] Chaumot A,Geffard O.Ecotoxicology, aquatic invertebrates.Encyclopedia of toxicology2014;2:284-8

[25] Parolini M.Toxicity of the Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) acetylsalicylic acid, paracetamol, diclofenac, ibuprofen and naproxen towards freshwater invertebrates: a review.Sci Total Environ2020;740:140043

[26] Mezzelani M,Da Ros Z.Ecotoxicological potential of non-steroidal anti-inflammatory drugs (NSAIDs) in marine organisms: bioavailability, biomarkers and natural occurrence in mytilus galloprovincialis.Mar Environ Res2016;121:31-9

[27] Branchet P,Piram A,Wong-Wah-Chung P.Pharmaceuticals in the marine environment: what are the present challenges in their monitoring?.Sci Total Environ2021;766:142644

[28] Świacka K,Kowalska D,Smolarz K.Presence of pharmaceuticals and their metabolites in wild-living aquatic organisms - Current state of knowledge.J Hazard Mat2022;424:127350

[29] Larsson DG,Paxeus N.Effluent from drug manufactures contains extremely high levels of pharmaceuticals.J Hazard Mater2007;148:751-5

[30] Majumder A,Ghosal PS.A review on hospital wastewater treatment: a special emphasis on occurrence and removal of pharmaceutically active compounds, resistant microorganisms, and SARS-CoV-2.J Environ Chem Eng2021;9:104812 PMCID:PMC7680650

[31] Hider-Mlynarz K,Maison P.Trends in analgesic consumption in France over the last 10 years and comparison of patterns across Europe.Br J Clin Pharmacol2018;84:1324-34 PMCID:PMC5980622

[32] Arpin-Pont L,Gomez E.Occurrence of PPCPs in the marine environment: a review.Environ Sci Pollut Res2016;23:4978-91

[33] Desbiolles F,Tiliacos C,Laffont-Schwob I.Occurrence and ecotoxicological assessment of pharmaceuticals: is there a risk for the Mediterranean aquatic environment?.Sci Total Environ2018;639:1334-48

[34] Ngubane NP,Ncube S,Madikizela LM.Determination of naproxen, diclofenac and ibuprofen in Umgeni estuary and seawater: a case of northern Durban in KwaZulu-Natal Province of South Africa.Reg Stud Mar Sci2019;29:100675

[35] Bonnefille B,Courant F,Fenet H.Diclofenac in the marine environment: a review of its occurrence and effects.Mar Pollut Bull2018;131:496-506

[36] Madikizela LM,Tutu H.Pharmaceuticals and their metabolites in the marine environment: sources, analytical methods and occurrence.Trends Environ Anal Chem2020;28:e00104

[37] Nödler K,Licha T.Polar organic micropollutants in the coastal environment of different marine systems.Mar Pollut Bull2014;85:50-9

[38] Borecka M,Haliński ŁP.Contamination of the southern Baltic Sea waters by the residues of selected pharmaceuticals: method development and field studies.Mar Pollut Bull2015;94:62-71

[39] Caban M,Lukaszewicz P.A new silylating reagent - dimethyl(3,3,3-trifluoropropyl)silyldiethylamine - for the derivatisation of non-steroidal anti-inflammatory drugs prior to gas chromatography-mass spectrometry analysis.J Chromatogr A2014;1346:107-16

[40] Szymczycha B,Białk-Bielińska A,Pazdro K.Submarine groundwater discharge as a source of pharmaceutical and caffeine residues in coastal ecosystem: bay of Puck, southern Baltic Sea case study.Sci Total Environ2020;713:136522

[41] Letsinger S,Rodríguez-Mozaz S,Barceló D.Spatial and temporal occurrence of pharmaceuticals in UK estuaries.Sci Total Environ2019;678:74-84

[42] Nebot C,Boyd KG.Quantification of human pharmaceuticals in water samples by high performance liquid chromatography-tandem mass spectrometry.Anal Chim Acta2007;598:87-94

[43] Vanryckeghem F,Van Langenhove H,Demeestere K.Multi-residue quantification and screening of emerging organic micropollutants in the Belgian Part of the North Sea by use of Speedisk extraction and Q-Orbitrap HRMS.Marine Pollution Bulletin2019;142:350-60

[44] Kallenborn R,Reiersen LO.Pharmaceuticals and personal care products (PPCPs) in Arctic environments: indicator contaminants for assessing local and remote anthropogenic sources in a pristine ecosystem in change.Environ Sci Pollut Res Int2018;25:33001-13

[45] González-Alonso S,Esteban S.Occurrence of pharmaceutical, recreational and psychotropic drug residues in surface water on the northern Antarctic Peninsula region.Environ Pollut2017;229:241-54

[46] McEneff G,Kelleher B,Quinn B.A year-long study of the spatial occurrence and relative distribution of pharmaceutical residues in sewage effluent, receiving marine waters and marine bivalves.Sci Total Environ2014;476-7:317-26

[47] Togola A.Multi-residue analysis of pharmaceutical compounds in aqueous samples.J Chromatogr A2008;1177:150-8

[48] Gonzalez-Rey M,Le Menach K,Budzinski H.Occurrence of pharmaceutical compounds and pesticides in aquatic systems.Mar Pollut Bull2015;96:384-400

[49] Lolić A,Santos LH,Correia M.Assessment of non-steroidal anti-inflammatory and analgesic pharmaceuticals in seawaters of North of Portugal: occurrence and environmental risk.Sci Total Environ2015;508:240-50

[50] Paíga P,Hellebuyck F,Correia M.Development of a SPE-UHPLC-MS/MS methodology for the determination of non-steroidal anti-inflammatory and analgesic pharmaceuticals in seawater.J Pharm Biomed Anal2015;106:61-70

[51] Baena-Nogueras RM,González-Mazo E.Determination of pharmaceuticals in coastal systems using solid phase extraction (SPE) followed by ultra performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS).Curr Anal Chem2016;12:183-201

[52] Maranho LA,Baena-Nogueras RM.Are WWTPs effluents responsible for acute toxicity? Seasonal variations of sediment quality at the Bay of Cádiz (SW, Spain).Ecotoxicology2015;24:368-80

[53] Afonso-Olivares C,Sosa-Ferrera Z.Assessment of the presence of pharmaceutical compounds in seawater samples from coastal area of Gran Canaria Island (Spain).Antibiotics2013;2:274-87 PMCID:PMC4790340

[54] Pintado-Herrera MG,Lara-Martín PA.Environmentally friendly analysis of emerging contaminants by pressurized hot water extraction-stir bar sorptive extraction-derivatization and gas chromatography-mass spectrometry.Anal Bioanal Chem2013;405:401-11

[55] Biel-Maeso M,Corada-Fernández C.Occurrence, distribution and environmental risk of pharmaceutically active compounds (PhACs) in coastal and ocean waters from the Gulf of Cadiz (SW Spain).Sci Total Environ2018;612:649-59

[56] Gros M,Barceló D.Fast and comprehensive multi-residue analysis of a broad range of human and veterinary pharmaceuticals and some of their metabolites in surface and treated waters by ultra-high-performance liquid chromatography coupled to quadrupole-linear ion trap tandem mass spectrometry.J Chromatogr A2012;1248:104-21

[57] Afsa S,Lara Martin PA.Occurrence of 40 pharmaceutically active compounds in hospital and urban wastewaters and their contribution to Mahdia coastal seawater contamination.Environ Sci Pollut Res Int2020;27:1941-55

[58] Brumovský M,Kohoutek J,Nizzetto L.Contaminants of emerging concern in the open sea waters of the Western Mediterranean.Environ Pollut2017;229:976-83

[59] Feo ML,Passoni A.Pharmaceuticals and other contaminants in waters and sediments from Augusta Bay (southern Italy).Sci Total Environ2020;739:139827

[60] Alygizakis NA,Borova VL,Hatzianestis I.Occurrence and spatial distribution of 158 pharmaceuticals, drugs of abuse and related metabolites in offshore seawater.Sci Total Environ2016;541:1097-105

[61] Nödler K,Aloupi M,Stasinakis AS.Evaluation of polar organic micropollutants as indicators for wastewater-related coastal water quality impairment.Environ Pollut2016;211:282-90

[62] Korkmaz NE,Aksu A,Caglar NB.Occurrence, sources and environmental risk assessment of pharmaceuticals in the Sea of Marmara, Turkey.Sci Total Environ2022;819:152996

[63] Korkmaz NE,Aksu A.Presence and distribution of selected pharmaceutical compounds in water and surface sediment of the Golden Horn Estuary, Sea of Marmara, Turkey.Reg Stud Mar Sci2022;51:102221

[64] Fang TH,Chin TS.The occurrence and distribution of pharmaceutical compounds in the effluents of a major sewage treatment plant in Northern Taiwan and the receiving coastal waters.Mar Pollut Bull2012;64:1435-44

[65] Sun Q,Li M.PPCPs in Jiulong River estuary (China): spatiotemporal distributions, fate, and their use as chemical markers of wastewater.Chemosphere2016;150:596-604

[66] Xie H,Xu N.Pharmaceuticals and personal care products in water, sediments, aquatic organisms, and fish feeds in the Pearl River Delta: Occurrence, distribution, potential sources, and health risk assessment.Sci Total Environ2019;659:230-9

[67] Ismail NAH,Haron DEM,Aris AZ.Occurrence of endocrine disrupting compounds in mariculture sediment of Pulau Kukup, Johor, Malaysia.Mar Pollut Bull2020;150:110735

[68] Wu J,Yang Z.Study on the matrix effect in the determination of selected pharmaceutical residues in seawater by solid-phase extraction and ultra-high-performance liquid chromatography-electrospray ionization low-energy collision-induced dissociation tandem mass spectrometry.J Chromatogr A2010;1217:1471-5

[69] Bayen S,Desai MM,Kelly BC.Occurrence and distribution of pharmaceutically active and endocrine disrupting compounds in Singapore’s marine environment: influence of hydrodynamics and physical-chemical properties.Environ Pollut2013;182:1-8

[70] Bayen S,Loh LL,Eikaas HS.Application of Polar Organic Chemical Integrative Sampler (POCIS) to monitor emerging contaminants in tropical waters.Sci Total Environ2014;482-3:15-22

[71] Bayen S,Juhel G,Kelly BC.Pharmaceutically active compounds and endocrine disrupting chemicals in water, sediments and mollusks in mangrove ecosystems from Singapore.Mar Pollut Bull2016;109:716-22

[72] Vidal-Dorsch DE,Maruya K,Trenholm RA.Contaminants of emerging concern in municipal wastewater effluents and marine receiving water.Environ Toxicol Chem2012;31:2674-82

[73] Comeau F,Brun GL.The occurrence of acidic drugs and caffeine in sewage effluents and receiving waters from three coastal watersheds in Atlantic Canada.Sci Total Environ2008;396:132-46

[74] Pereira CDS,Cortez FS.Occurrence of pharmaceuticals and cocaine in a Brazilian coastal zone.Sci Total Environ2016;548-9:148-54

[75] Fontes MK,Maranho LA.A tiered approach to assess effects of diclofenac on the brown mussel Perna perna: a contribution to characterize the hazard.Water Res2018;132:361-70

[76] Weigel S,Jensen E,Thoresen H.Determination of selected pharmaceuticals and caffeine in sewage and seawater from Tromsø/Norway with emphasis on ibuprofen and its metabolites.Chemosphere2004;56:583-92

[77] Brumovský M,Kohoutek J.Exploring the occurrence and distribution of contaminants of emerging concern through unmanned sampling from ships of opportunity in the North Sea.J Mar Syst2016;162:47-56

[78] Togola A.Analytical development for analysis of pharmaceuticals in water samples by SPE and GC-MS.Anal Bioanal Chem2007;388:627-35

[79] Loos R,Paracchini B,Weissteiner C.Analysis of polar organic contaminants in surface water of the northern Adriatic Sea by solid-phase extraction followed by ultrahigh-pressure liquid chromatography-QTRAP® MS using a hybrid triple-quadrupole linear ion trap instrument.Anal Bioanal Chem2013;405:5875-85

[80] Jiang JJ,Fang MD.Emerging organic contaminants in coastal waters: anthropogenic impact, environmental release and ecological risk.Mar Pollut Bull2014;85:391-9

[81] Chen H,Guo H,Zhang Y.Pharmaceuticals and personal care products in the seawater around a typical subtropical tourist city of China and associated ecological risk.Environ Sci Pollut Res Int2021;28:22716-28 PMCID:PMC7797026

[82] Chen WL,Lee DJH,Chen ZY.Targeted profiling of chlorinated transformation products and the parent micropollutants in the aquatic environment: a comparison between two coastal cities.Chemosphere2020;242:125268

[83] Waleng NJ.Occurrence of pharmaceuticals in the environmental waters: African and Asian perspectives.Environ Chem Ecotoxicol2022;4:50-66

[84] Long ER,Weakland S,Benskin JP.Quantification of pharmaceuticals, personal care products, and perfluoroalkyl substances in the marine sediments of Puget Sound, Washington, USA.Environ Toxicol Chem2013;32:1701-10

[85] Postigo C,López-García E,López de Alda M.Human footprint on the water quality from the northern Antarctic Peninsula region.J Hazard Mater2023;453:131394

[86] Ridgway J.Estuaries as repositories of historical contamination and their impact on shelf seas.Estuar Coast Shelf Sci2002;55:903-28

[87] Moreno-González R,Gros M,Barceló D.Input of pharmaceuticals through coastal surface watercourses into a Mediterranean lagoon (Mar Menor, SE Spain): sources and seasonal variations.Sci Total Environ2014;490,59-72

[88] Rogers HR.Sources, behaviour and fate of organic contaminants during sewage treatment and in sewage sludges.Sci Total Environ1996;185:3-26

[89] Fent K.Progestins as endocrine disrupters in aquatic ecosystems: concentrations, effects and risk assessment.Environ Int2015;84:115-30

[90] Mezzelani M,Fattorini D.Transcriptional and cellular effects of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) in experimentally exposed mussels, Mytilus galloprovincialis.Aquat Toxicol2016;180:306-19

[91] Courant F,Bonnefille B.Exposure of marine mussels to diclofenac: modulation of prostaglandin biosynthesis.Environ Sci Pollut Res Int2018;25:6087-94

[92] Freitas R,Costa S.Does salinity modulates the response of Mytilus galloprovincialis exposed to triclosan and diclofenac?.Environ Pollut2019;251:756-65

[93] Freitas R,Costa S.The influence of temperature on the effects induced by Triclosan and Diclofenac in mussels.Sci Total Environ2019;663:992-9

[94] Mezzelani M,Fattorini D.Long-term exposure of Mytilus galloprovincialis to diclofenac, Ibuprofen and Ketoprofen: Insights into bioavailability, biomarkers and transcriptomic changes.Chemosphere2018;198:238-48

[95] Álvarez-Ruiz R,Campo J.Bioaccumulation of emerging contaminants in mussel (Mytilus galloprovincialis): influence of microplastics.Sci Tot Environ2021;796:149006

[96] Bonnefille B,Gomez E,Courant F.Metabolic profiling identification of metabolites formed in Mediterranean mussels (Mytilus galloprovincialis) after diclofenac exposure.Sci Total Environ2017;583:257-68

[97] Ericson H,Kumblad L.Physiological effects of diclofenac, ibuprofen and propranolol on Baltic Sea blue mussels.Aquat Toxicol2010;99:223-31

[98] Świacka K,Caban M.Evaluation of bioconcentration and metabolism of diclofenac in mussels Mytilus trossulus - laboratory study.Mar Pollut Bull2019;141:249-55

[99] Costa S,Pretti C.The influence of climate change related factors on the response of two clam species to diclofenac.Ecotoxicol Environ Saf2020;189:109899

[100] Serrano MA,Mattos JJ.Differential gene transcription, biochemical responses, and cytotoxicity assessment in Pacific oyster Crassostrea gigas exposed to ibuprofen.Environ Sci Pollut Res Int2015;22:17375-85

[101] Mello FV,Fogaça FHS,Torres JPM.Occurrence of pharmaceuticals in seafood from two Brazilian coastal areas: implication for human risk assessment.Sci Total Environ2022;803:149744

[102] Cunha SC,Fernandes JO.Mussels as bioindicators of diclofenac contamination in coastal environments.Environ Pollut2017;225:354-60

[103] Mezzelani M,Gorbi S,Regoli F.Human pharmaceuticals in marine mussels: evidence of sneaky environmental hazard along Italian coasts.Mar Environ Res2020;162:105137

[104] Capolupo M,Kiwan A.A comprehensive evaluation of the environmental quality of a coastal lagoon (Ravenna, Italy): integrating chemical and physiological analyses in mussels as a biomonitoring strategy.Sci Total Environ2017;598:146-59

[105] Wolecki D,Pazdro K,Stepnowski P.Simultaneous determination of non-steroidal anti-inflammatory drugs and natural estrogens in the mussels Mytilus edulis trossulus.Talanta2019;200:316-23

[106] Omar TFT,Yusoff FM.Occurrence and level of emerging organic contaminant in fish and mollusk from Klang River estuary, Malaysia and assessment on human health risk.Environ Pollut2019;248:763-73

[107] Cravo A,Rodriguez J.Understanding the bioaccumulation of pharmaceutical active compounds by clams Ruditapes decussatus exposed to a UWWTP discharge.Environ Res2022;208:112632

[108] Granek EF,Nilsen EB.Spatial and temporal variability of contaminants within estuarine sediments and native Olympia oysters: a contrast between a developed and an undeveloped estuary.Sci Tot Environ2016;557-8:869-79

[109] Commission Regulation (EU) No 253/2011 of 15 March 2011 amending Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) as regards Annex XIII Text with EEA relevance. Available from: https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=celex%3A32011R0253. [Last accessed on 2 Aug 2023]

[110] Guideline on the environmental risk assessment of medicinal products for human use. European Medicines Agency, Committee for Medicinal Products for Human Use (CHMP). Available from: https://www.ema.europa.eu/en/documents/scientific-guideline/draft-guideline-environmental-risk-assessment-medicinal-products-human-use-revision-1_en.pdf. [Last accessed on 2 Aug 2023]

[111] Freitas R,Pires A.The effects of carbamazepine on macroinvertebrate species: comparing bivalves and polychaetes biochemical responses.Water Res2015;85:137-47

[112] Contardo-Jara V,Pflugmacher S,Kloas W.Molecular effects and bioaccumulation of levonorgestrel in the non-target organism Dreissena polymorpha.Environ Pollut2011;159:38-44

[113] Beyer J,Brooks S.Blue mussels (Mytilus edulis spp.) as sentinel organisms in coastal pollution monitoring: a review.Mar Environ Res2017;130:338-65

[114] Miller TH,Owen SF,Barron LP.A review of the pharmaceutical exposome in aquatic fauna.Environ Pollut2018;239:129-46 PMCID:PMC5981000

[115] UNESCO and HELCOM. Pharmaceuticals in the aquatic environment of the Baltic Sea region: a status report. Available from: https://www.helcom.fi/wp-content/uploads/2019/08/BSEP149.pdf. [Last accessed on 4 Aug 2023]

[116] Eades C.The effects of diclofenac on the physiology of the green shore crab Carcinus maenas.Mar Environ Res2010;69 Suppl:S46-8

[117] González-Ortegón E,Le Vay L.A multiple stressor approach to study the toxicity and sub-lethal effects of pharmaceutical compounds on the larval development of a marine invertebrate.J Hazard Mater2013;263 Pt 1:233-8

[118] Pérez S,Beiras R.Acute toxicity of selected organic pollutants to saltwater (mysid Siriella armata) and freshwater (cladoceran Daphnia magna) ecotoxicological models.Ecotoxicology2015;24:1229-38

[119] Trombini C,Blasco J.Evaluation of acute effects of four pharmaceuticals and their mixtures on the copepod Tisbe battagliai.Chemosphere2016;155:319-28

[120] Schmidt W,Hernan R.Effects of the pharmaceuticals gemfibrozil and diclofenac on the marine mussel (Mytilus spp.) and their comparison with standardized toxicity tests.Mar Pollut Bull2011;62:1389-95

[121] Mauro M,Celi M.Effects of diclofenac on the gametes and embryonic development of Arbacia lixula.Eur Zool J2022;89:535-45

[122] Zanuri NB, Bentley MG, Caldwell GS. Assessing the impact of diclofenac, ibuprofen and sildenafil citrate (Viagra^®) on the fertilisation biology of broadcast spawning marine invertebrates.Mar Environ Res2017;127:126-36

[123] Ribeiro S,Martins R.Toxicity screening of diclofenac, propranolol, sertraline and simvastatin using Danio rerio and Paracentrotus lividus embryo bioassays.Ecotoxicol Environ Saf2015;114:67-74

[124] Schmidt W,McEneff G,Quinn B.A proteomic evaluation of the effects of the pharmaceuticals diclofenac and gemfibrozil on marine mussels (Mytilus spp.): evidence for chronic sublethal effects on stress-response proteins.Drug Test Anal2014;6:210-9

[125] Jaafar SN,Sheehan D.Redox proteomic analysis of mytilus edulis gills: effects of the pharmaceutical diclofenac on a non-target organism.Drug Test Anal2015;7:957-66

[126] Balbi T,Fabbri R.Diclofenac affects early embryo development in the marine bivalve Mytilus galloprovincialis.Sci Total Environ2018;642:601-9

[127] Fabbri R,Balbi T,Palumbo F.Adaptation of the bivalve embryotoxicity assay for the high throughput screening of emerging contaminants in Mytilus galloprovincialis.Mar Environ Res2014;99:1-8

[128] Gonzalez-Rey M.Effects of non-steroidal anti-inflammatory drug (NSAID) diclofenac exposure in mussel Mytilus galloprovincialis.Aquat Toxicol2014;148:221-30

[129] Bonnefille B,Alali M,Fenet H.Metabolomics assessment of the effects of diclofenac exposure on Mytilus galloprovincialis: Potential effects on osmoregulation and reproduction.Sci Total Environ2018;613-4:611-8

[130] Toufexi E,Vlastos D.Mediated effect of ultrasound treated Diclofenac on mussel hemocytes: first evidence for the involvement of respiratory burst enzymes in the induction of DCF-mediated unspecific mode of action.Aquat Toxicol2016;175:144-53

[131] Świacka K,Maculewicz J.Effects of environmentally relevant concentrations of diclofenac in Mytilus trossulus.Sci Total Environ2020;737:139797

[132] Świacka K,Świeżak J,Smolarz K.A multi-biomarker approach to assess toxicity of diclofenac and 4-OH diclofenac in Mytilus trossulus mussels - First evidence of diclofenac metabolite impact on molluscs.Environ Pollut2022;315:120384

[133] Trombini C,Blasco J.Assessing the effect of human pharmaceuticals (carbamazepine, diclofenac and ibuprofen) on the marine clam Ruditapes philippinarum: an integrative and multibiomarker approach.Aquat Toxicol2019;208:146-56

[134] Nunes B,Canelas GG,Correia AT.Toxic effects of environmentally realistic concentrations of diclofenac in organisms from two distinct trophic levels, Hediste diversicolor and Solea senegalensis.Comp Biochem Physiol C Toxicol Pharmacol2020;231:108722

[135] Maranho LA,Baena-Nogueras RM,DelValls TA.A candidate short-term toxicity test using Ampelisca brevicornis to assess sublethal responses to pharmaceuticals bound to marine sediments.Arch Environ Contam Toxicol2015;68:237-58

[136] Aguirre-Martínez GV,Fabbri E,Martín-Díaz ML.Stability of lysosomal membrane in Carcinus maenas acts as a biomarker of exposure to pharmaceuticals.Environ Monit Assess2013;185:3783-93

[137] Aguirre-Martínez GV,Martín-Díaz ML.Identification of biomarkers responsive to chronic exposure to pharmaceuticals in target tissues of Carcinus maenas.Mar Environ Res2013;87-8:1-11

[138] Pusceddu FH,Pereira CDS.Environmental risk assessment of triclosan and ibuprofen in marine sediments using individual and sub-individual endpoints.Environ Pollut2018;232:274-83

[139] Aguirre-Martínez GV,Garrido-Pérez C,Del Valls TA.Are standard tests sensitive enough to evaluate effects of human pharmaceuticals in aquatic biota? Facing changes in research approaches when performing risk assessment of drugs.Chemosphere2015;120:75-85

[140] Gonzalez-Rey M.Non-steroidal anti-inflammatory drug (NSAID) ibuprofen distresses antioxidant defense system in mussel Mytilus galloprovincialis gills.Aquat Toxicol2011;105:264-9

[141] Maria VL,Bebianno MJ.Transcriptomic effects of the non-steroidal anti-inflammatory drug Ibuprofen in the marine bivalve Mytilus galloprovincialis Lam.Mar Environ Res2016;119:31-9

[142] Aguirre-Martínez GV,Martín-Díaz ML.General stress, detoxification pathways, neurotoxicity and genotoxicity evaluated in Ruditapes philippinarum exposed to human pharmaceuticals.Ecotoxicol Environ Saf2016;124:18-31

[143] Aguirre-Martínez GV,Fabbr E,Martín-Díaz ML.Using lysosomal membrane stability of haemocytes in Ruditapes philippinarum as a biomarker of cellular stress to assess contamination by caffeine, ibuprofen, carbamazepine and novobiocin.J Environ Sci2013;25:1408-18

[144] Milan M,Patarnello T,Marin MG.Gene transcription and biomarker responses in the clam Ruditapes philippinarum after exposure to ibuprofen.Aquat Toxicol2013;126:17-29

[145] Matozzo V,Marin MG.The nonsteroidal anti-inflammatory drug, ibuprofen, affects the immune parameters in the clam Ruditapes philippinarum.Mar Environ Res2012;79:116-21

[146] Maranho LA,Lara-Martín PA,Martín-Díaz ML.Bioavailability, oxidative stress, neurotoxicity and genotoxicity of pharmaceuticals bound to marine sediments. The use of the polychaete Hediste diversicolor as bioindicator species.Environ Res2014;134:353-65

[147] Maranho LA,DelValls TA,Martín-Díaz ML.Toxicological evaluation of sediment samples spiked with human pharmaceutical products: energy status and neuroendocrine effects in marine polychaetes Hediste diversicolor.Ecotoxicol Environ Saf2015;118:27-36

[148] CEC (Commission of the European Communities), 1996. Part II. Available from: https://echa.europa.eu/documents/10162/987906/tgdpart2_2ed_en.pdf/138b7b71-a069-428e-9036-62f4300b752f. [Last accessed on 2 Aug 2023]

[149] Almeida A,Soares AMVM.Anti-inflammatory drugs in the marine environment: bioconcentration, metabolism and sub-lethal effects in marine bivalves.Environ Pollut2020;263:114442

[150] Lotterhos KE. Gamete release and spawning behavior in broadcast spawning marine invertebrates. Available from: https://www.researchgate.net/publication/312449344. [Last accessed on 2 Aug 2023]

[151] Pereira CDS,Choueri RB.Ecological relevance of Sentinels’ biomarker responses: a multi-level approach.Mar Environ Res2014;96:118-26

[152] Rowley AF,Taylor GW.Prostaglandins in non-insectan invertebrates: recent insights and unsolved problems.J Exp Biol2005;208:3-14

[153] Munari M,Finos L,Giani M.Coping with seawater acidification and the emerging contaminant diclofenac at the larval stage: a tale from the clam Ruditapes philippinarum.Chemosphere2016;160:293-302

[154] Regoli F.Oxidative pathways of chemical toxicity and oxidative stress biomarkers in marine organisms.Mar Environ Res2014;93:106-17

[155] Winston GW.Prooxidant and antioxidant mechanisms in aquatic organisms.Aquatic Toxicology1991;19:137-61

[156] Leone S,Bertolini A.Dual acting anti-inflammatory drugs.Curr Top Med Chem2007;7:265-75

[157] Bertolini A,Sandrini M.Selective COX-2 inhibitors and dual acting anti-inflammatory drugs: critical remarks.Curr Med Chem2002;9:1033-43

[158] Technical guidance document on risk assessment. In support of Commission directive 93/67/EEC on risk assessment for new notified substances, Commission Regulation (EC) No 1488/94 on risk assessment for existing substances, Directive 98/8/EC of the European Parliament and of the council concerning the placing of biocidal products on the market. Part I. Available from: https://echa.europa.eu/documents/10162/16960216/tgdpart1_2ed_en.pdf/f0fb9a44-13c9-44d7-897f-f7b3fe8ccacb. [Last accessed on 2 Aug 2023]

[159] Beiras R.Environmental risk assessment of pharmaceutical and personal care products in estuarine and coastal waters. In: Durán-Álvarez JC, Jiménez-Cisneros B, editors. Pharmaceuticals in Marine and Coastal Environments. Elsevier; 2021. p. 195-252.

[160] Durán I.Acute water quality criteria for polycyclic aromatic hydrocarbons, pesticides, plastic additives, and 4-Nonylphenol in seawater.Environ Pollut2017;224:384-91

[161] Report of the OECD Workshop on the extrapolation of laboratory aquatic toxicity data on the real environment. Organization for Economic Cooperation and Development (OECD), OECD Environment Monographs 1992; No.59, Paris. Available from: https://www.oecd.org/chemicalsafety/testing/34528236.pdf. [Last accessed on 2 Aug 2023]

[162] Hansen P.Risk assessment of emerging contaminants in aquatic systems.TrAC Trends Anal Chem2007;26:1095-9

[163] Kötke D,Xie Z.Prioritised pharmaceuticals in German estuaries and coastal waters: occurrence and environmental risk assessment.Environ Pollut2019;255:113161

[164] DeLorenzo ME.Individual and mixture effects of selected pharmaceuticals and personal care products on the marine phytoplankton species Dunaliella tertiolecta.Arch Environ Contam Toxicol2008;54:203-10

[165] Minguez L,Farcy E,Budzinski H.Toxicities of 48 pharmaceuticals and their freshwater and marine environmental assessment in northwestern France.Environ Sci Pollut Res2016;23:4992-5001

[166] Kuzmanović M,Petrović M.Risk assessment based prioritization of 200 organic micropollutants in 4 Iberian rivers.Sci Total Environ2015;503-4:289-99

[167] Manual on the methodological framework to derive environmental quality standards for priority substances. Available from: https://www.helpdeskwater.nl/@177379/manual-environmental/. [Last accessed on 2 Aug 2023]

[168] Posthuma L,Traas TP. General introduction to species sensitivity distribution. In: Posthuma L, Traas TP, Suter II GW, editors. Species sensitivity distributions in ecotoxicology. CRC, Boca Ratón; 2002. p. 1-10. Available from: http://www.ievbras.ru/ecostat/Kiril/R/Ecotox/Leo%20Posthuma,%20Glenn%20W.%20Suter%20II,%20Theo%20P.%20Traas-Species%20Sensitivity%20Distributions%20in%20Ecotoxicology-CRC%20Press%20(2001).pdf. [Last accessed on 2 Aug 2023]

[169] Yanagihara M,Iwasaki Y.Can chemical toxicity in saltwater be predicted from toxicity in freshwater? A comprehensive evaluation using species sensitivity distributions.Environ Toxicol Chem2022;41:2021-7 PMCID:PMC9542858

[170] Piva F,Onorati F.Assessing sediment hazard through a weight of evidence approach with bioindicator organisms: a practical model to elaborate data from sediment chemistry, bioavailability, biomarkers and ecotoxicological bioassays.Chemosphere2011;83:475-85

[171] Benedetti M,Piva F.A multidisciplinary weight of evidence approach for classifying polluted sediments: Integrating sediment chemistry, bioavailability, biomarkers responses and bioassays.Environ Int2012;38:17-28

[172] Larsen JC. Basic concepts and terminology used to describe the combined action of chemicals in mixtures. In: Combined actions and interactions of chemicals mixtures. The toxicological effects of exposure to mixture of industrial and environmental chemicals. FødevareRapport; 2003. p. 20-32. Available from: https://www.food.dtu.dk/english/-/media/institutter/foedevareinstituttet/publikationer/pub-2003/combined-actions-and-interactions-of-chemicals-in-mixture.pdf?la=da&hash=1B5BD41C9DE06EEABABBAEC6124EF2C9C9FEFC72. [Last accessed on 2 Aug 2023]

[173] Rodea-Palomares I,Martín-Betancor K,Fernández-Piñas F.Additivity and interactions in ecotoxicity of pollutant mixtures: some patterns, conclusions, and open questions.Toxics2015;3:342-69 PMCID:PMC5606646

[174] Kortenkamp A,Faust M. State of the art report on mixture toxicity. Final report, Executive summary 2009. Available from: https://www.pan-europe.info/old/Campaigns/pesticides/documents/cum_syn_effects/Kortenkamp%20state%20of%20the%20art%20mixture%20toxicity.pdf. [Last accessed on 2 Aug 2023]

[175] Schnell S,Barata C.Single and combined toxicity of pharmaceuticals and personal care products (PPCPs) on the rainbow trout liver cell line RTL-W1.Aquat Toxicol2009;93:244-52

[176] Coors A,Schoknecht U,Kehrer A.Predicting acute and chronic effects of wood preservative products in Daphnia magna and Pseudokirchneriella subcapitata based on the concept of concentration addition.Environ Toxicol Chem2014;33:382-93

[177] Coors A,Heim J,Kehrer A.Environmental risk assessment of biocidal products: identification of relevant components and reliability of a component-based mixture assessment.Environ Sci Eur2018;30:3 PMCID:PMC5773622

[178] Puckowski A,Wagil M.Mixture toxicity of flubendazole and fenbendazole to Daphnia magna.Int J Hyg Environ Health2017;220:575-82

[179] Sigurnjak M,Cvetnić M.Combined toxicities of binary mixtures of alachlor, chlorfenvinphos, diuron and isoproturon.Chemosphere2020;240:124973

[180] Backhaus T.Predictive environmental risk assessment of chemical mixtures: a conceptual framework.Environ Sci Technol2012;46:2564-73

[181] UK Environment Agency (Gov UK). The direct toxicity assessment of aqueous environmental samples using the marine copepod Tisbe battagliai lethality test (2007). Methods for the Examination of Waters and Associated Materials. Available from: https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/755574/tisbe210_jan26_1496121.pdf. [Last accessed on 2 Aug 2023]

[182] Gonzalez-Rey M,Piazza CE,Bebianno MJ.Effects of active pharmaceutical ingredients mixtures in mussel Mytilus galloprovincialis.Aquat Toxicol2014;153:12-26

[183] Trombini C,Villar-Navarro M.Bioaccumulation and biochemical responses in the peppery furrow shell Scrobicularia plana exposed to a pharmaceutical cocktail at sub-lethal concentrations.Ecotoxicol Environ Saf2022;242:113845

[184] Fabbri R,Balbi T,Palumbo F.Adaptation of the bivalve embryotoxicity assay for the high throughput screening of emerging contaminants in Mytilus galloprovincialis.Mar Environ Res2014;99:1-8

[185] Luna-Acosta A,Thomas-Guyon H.Detection of early effects of a single herbicide (diuron) and a mix of herbicides and pharmaceuticals (diuron, isoproturon, ibuprofen) on immunological parameters of Pacific oyster (Crassostrea gigas) spat.Chemosphere2012;87:1335-40

[186] Munari M,Gagné F.Does exposure to reduced pH and diclofenac induce oxidative stress in marine bivalves? A comparative study with the mussel Mytilus galloprovincialis and the clam Ruditapes philippinarum.Environ Pollut2018;240:925-37

[187] Ankley GT,Erickson RJ.Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment.Environ Toxicol Chem2010;29:730-41

[188] Dumas T,Gomez E.Metabolomics approach reveals disruption of metabolic pathways in the marine bivalve Mytilus galloprovincialis exposed to a WWTP effluent extract.Sci Total Environ2020;712:136551