Ecotoxicology https://doi.org/10.1007/s10646-021-02507-1 Status of pharmaceuticals in the Korle Lagoon and their toxicity to non-target organisms Ebenezer Aquisman Asare 1,2 Accepted: 19 November 2021 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021 Abstract The availability of pharmaceutically active compounds (PhACs) in surface waters and suspended solids/sediments presents an ecological hazard of chronic exposure to non-target organisms. Thus, water and sediment samples were collected from the Korle Lagoon in the west of Accra-Ghana city center to evaluate 35 medicinal drugs belonging to the main therapeutic classes and their toxicity to non-target organisms (i.e., fish, daphnid, and algae). High-performance liquid chromatography coupled to mass spectrometry (HPLC-MS/MS) was employed to analyze the levels of PhACs in the samples. PhACs levels in water samples were higher compared to PhACs levels in sediment samples. Acetaminophen, ibuprofen, tramadol, and Diclofenac were the PhACs that showed a higher frequency of detections and higher average concentrations. Diazepam, mefenamic acid, indomethacin, gemfibrozil, and glibenclamide exhibited a higher frequency of detections, but their average concentrations in both sample types were lower. The calculated risk index values for acetaminophen and ibuprofen suggested low ecological risks to fish, while tramadol showed medium to high ecological risks to daphnid. In contrast, acetaminophen and fenofibrate showed low ecological risks to daphnid. Additionally, the risk index values for fenofibrate suggested medium to high ecological risks to algae, while tramadol exhibited low ecological risks to algae. The other PhACs showed negligible ecological risks to non-target organisms. The calculated toxic unit values for each sampled site suggested a medium adverse ecological risk to non-target organisms. Based on the results obtained, the availability of PhACs in the studied area will have adverse effects on studied non-target organisms. The negative impacts of PhACs on non-target organisms may cause an imbalance in the food chain process, leading to a decrease in fish production and a reduction in fish quality. The result of this study is evidence of public health threat because the accumulation of PhACs in fish species may also cause some kinds of hormonal, chemical, and molecular changes within the various systems of the fishes to be toxic or unpleasant for humans’ consumption. Keywords Pharmaceutically active compounds ● Eco-toxicological data ● Environmental risk assessment ● Lethal concentration ● Effect concentration ● Korle Lagoon Introduction In addition to the primary contaminants such as heavy metals, polychlorinated biphenyls, pesticides, aliphatic and Supplementary information The online version contains polycyclic aromatic hydrocarbons, etc. which are threaten- supplementary material available at https://doi.org/10.1007/s10646- ing the environment, the so-called unregulated emerging 021-02507-1. pollutants are gaining grounds in environmental con- * Ebenezer Aquisman Asare tamination (da Silva et al. 2011; Asare 2016; Molnar et al. aquisman1989@gmail.com 2020; Asare 2021). Emerging contaminants are artificial or naturally occurring chemicals or microbes that are not 1 Department of Chemistry, Faculty of Resource Science and regularly monitored in the environment but can enter and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia result in known or believed unfavorable environmental 2 effects (da Silva et al. 2011). Generally, these chemicalsDepartment of Nuclear Science and Applications, Graduate School of Nuclear and Allied Sciences, University of Ghana, AE1 manufactured commercially have many uses and are Kwabenya-Accra, Ghana essential to our present-day society. Among these numerous 1234567890();,: 1234567890();,: E. A. Asare emerging contaminants, pharmaceuticals are finding their of national rivers or lagoons for the investigation of PhACs. way into the entire ecological compartment. One of the coastal wetlands in Ghana is Korle Lagoon Pharmaceuticals are different classes of compounds which consists of an open lagoon, salts pans, marsh, designed to cure or treat illness and enhance health. Phar- beautiful dunes, and scrubs. It is in the north-eastern portion maceuticals have improved in recent decades, promoting of Agbogbloshie town in which one of Ghana’s largest the increase of mean age, growth and well-being of people dumping sites is located. The lagoon flows from the west (Guzel et al. 2019). The usage of pharmaceuticals has center of Accra and joins the coast of the Gulf of Guinea. In increased remarkably. Technologies for wastewater treat- the 1990s, the administration of Ghana set up the Korle ment are not feasible and efficient for detaching all sorts of Lagoon ecological restoration project, intending to reinstate pharmaceutically active compounds (PhACs) with the same the lagoon to a more natural condition and lessen con- competence. Thus, most PhACs with their metabolites and tamination (Boadi and Kuitunen 2002), but an unplanned conjugates have emerged in every corner of ecological encampment, Old Fadama, was created by some 70,000 partitions (i.e., biota, surface waters, sediment, etc.) (Hal- citizens on the banks of the lagoon. The current state of the ling-Sorensen et al., (1998); Kummerer 2004; Molnar et al. lagoon is an eyesore. The lagoon is polluted with different 2020). The study of medicine’s effects on humans is forms of waste by the influence of anthropogenic activities. through safety and toxicology research; the likely ecological Furthermore, researchers focused only on heavy metals impacts of their presentation and usage are not much in and around the lagoon (Boadi and Kuitunen 2002; Fosu- recognized and have raised an issue of concern (Boxall Mensah et al. 2017), but information regarding PhACs 2004). In addition, knowledge regarding the potential levels in water and surface sediments of the lagoon and their adverse health impacts of PhACs on non-target organisms toxicity to non-target organisms are lacking. For these such as phytoplankton, zooplankton, crustaceans, mollusks, reasons, this work aimed to examine the status and envir- and fish when the various PhACs combine in complex onmental risk assessment of PhACs in water and surface mixtures in aquatic environments is inadequate (Guzel et al. sediments collected from the Korle Lagoon. Hence, treated 2019). Komori et al. opined that quantification and hazard samples were analyzed to evaluate 35 pharmaceuticals evaluation of PhACs in the environment is on individual belonging to the various therapeutic classes such as anti- compounds (Komori et al. 2013). However, PhACs do not diabetics, psychiatric drugs, antibiotics, analgesics, and always occur as isolated matter in the environment (Komori anti-inflammatory drugs, ß-blockers, anti-ulcer agent, et al. 2013; Molnar et al. 2020). Thus, to get a logical cholesterol-lowering statin, lipid regulator drugs, and overview of environment involvement and exploration, diuretics and their potential risk to non-target organisms evaluation of multicomponent mixture effect of PhACs is (i.e., fish, daphnid, and algae). needed (Heys et al., (2016)). Also, the actual elucidation of measured environmental concentration (MEC) of PhACs is a problem for researchers (Guzel et al. 2019). The avail- Materials and methods ability of data on empirical toxicity of PhACs such as no observed effect concentration (NOEC), median effective Chemicals concentration (EC50), and median lethal concentration (LC50) is lacking, and on condition that such information is Pharmaceutical standards with a high purity grade (>90%) available; their descriptions are from different observations. were used to identify target compounds. Diclofenac, For example, other species and different endpoints, so, that naproxen, gemfibrozil, ibuprofen, and ketoprofen were is to say, they are inconsistent (Guzel et al. 2019). A hazard purchased from Jesuder, Rubi, Spain. Butalbital, lorazepam, quotient (RQ) is applied to evaluate the potential risk of and diazepam were supplied by Cerilliant, Texas, USA. PhACs in any environmental media. RQ is the rate of the Atorvastatin was purchased from LGC Promochem, Lon- maximum MEC to the predicted no-effect concentrations don, UK. Mevastatin, indomethacin, phenazone, acet- (PNEC). In this context, the PNEC depends on the available aminophen, fenofibrate, mefenamic acid, famotidine, toxicological data (Komori et al. 2013; Molnar et al. 2020). bezafibrate, cimetidine (hydrochloride), josamycin, azi- In recent times, Ghana has raised its stand in the African thromycin (dehydrate), ranitidine (hydrochloride), tyrosine pharmaceutical market and utilization (Ghana Pharmaceu- A., carbamazepine, tramadol, chloramphenicol, metronida- tical report 2012). Such high utilization may result in the zole, erythromycin (hydrate), clarithromycin, pravastatin verdict that the problem relating to aquatic pollution by (sodium salt), trimethoprim, furosemide, nadolol, glib- PhACs could be a critical concern, and thus PhACs in water enclamide, atenolol, and hydrochlorothiazide were received bodies must be evaluated. Additionally, because data are from Sigma-Aldrich, Steinheim, Germany. unavailable on the pollution levels of PhACs in Ghanaian The internal standards (IS) used were isotopically labeled aquatic ecosystems, it is necessary to establish a hierarchy compounds. They include erythromycin-13C,d 133 (N-Methyl- C, Status of pharmaceuticals in the Korle Lagoon and their toxicity to non-target organisms d3), diclofenac-d4, rac-naproxen-d3, gemfibrozil-d6, ibuprofen- sampler was used to collect the surface sediments (0–5 cm). d3, ketoprofen- 13CD3, diazepam-d5, acetaminophen-d4, Ten surface sediment samples were grabbed, placed in atorvastatin-d5 sodium salt, pravastatin-d3, metronidazole polyethylene bags, and kept in an ice chest at a temperature hydroxyl-d2, bezafibrate-d4, hydrochlorothiazide-3,3-d2, and of 50 °C. The samples were transported to the laboratory fenofibrate-d6 from Ghana Food and Drugs Authority. Sol- within 48 h. In the laboratory, sediment samples were pre- vents, acetonitrile, HPLC grade methanol, formic acid, and served at 15 °C and later freeze-dried for 72 h. The freeze- water (Lichrosolv) were provided by Merck, Darmstadt, Ger- dried samples were ground using mortar and pestle, sieved many. Sankofa Ghana Air Liquid supplied nitrogen gas through a 125 μm plastic sieve, and homogenized. The (99.995% purity) for drying. powdery samples were stored in pre-cleaned plastic bottles in a refrigerator at 25 °C before extraction. Sample collection Preparation of standard solutions Korle Lagoon is 104 m above sea level with the coordinate 5°33'0″N and 0°13'0″W. Water and surface sediment sam- Each standard solution and isotopically labeled internal ples were collected in February 2021 (Fig. 1). For easy standard solutions were prepared based on mass in identification, samples were labeled based on the type of CH3OH. Diazepam and lorazepam were dissolved in sample medium collected. That is, KLW signifies water CH3OH at a concentration of 1 mg/mL. Furosemide was samples and KLS denotes surface sediment samples. The obtained as a solution in acetonitrile. The solutions were protocol for collecting water samples was adapted by kept in a refrigerator at 25 °C. Because of the limited Molnar et al. (2020). Water-column sample devices were stability of fresh stock solutions of antibiotics, they were used to collect the water samples from the middle of the prepared monthly. The rest of the stock solutions were water in 1.5 L glass bottles with Teflon-faced caps. A total restored every 120 days. All medicines were prepared of 10 samples were collected from 10 sampling sites. A pure using a suitable dilution factor for each stock solution in formic acid was used to acidify each liter of sample to a mixture of CH3OH and H2O (1:3, v/v), and after each reduce the pH to 3.5–4.0 due to sorbent type compatibility. analytical run, they were renewed. Also, a different Samples were stored in an ice chest at a temperature of 6 °C combination of an isotopically labeled internal standard and transported to the laboratory within 72 h. used for internal standard measurement was prepared in The protocol for collecting surface sediments from the CH3OH and then diluted in a mixture of CH3OH and lagoon was adapted by da Silva et al. (2011). A grab H2O (1:3, v/v). Fig. 1 Satellite map of the study area showing sampled sites KL1 KL2 KL3 KL4 KL6 KL5 KL7 KL8 KL9 KL10 E. A. Asare Sample preparation Environmental risk assessment (ERA) Techniques for water and surface sediment samples pre- Predicted no-effect concentrations (PNEC) paration before analyses were adapted by Gros et al. (2009). Environmental risk assessment is dependent on eco- Surface sediment samples toxicological threshold data from experiments on aquatic organisms (fish species, daphnid, and or algae). Thus, the Approximately 1.5 g of surface sediment sample was mea- values for NOEC and E(L)C50 obtained from chronic and sured and extracted using the pressurized liquid extraction acute tests, respectively, are considered. Applying the technique. The extraction solvents (i.e., a mixture of NOEC and E(L)C50 values, the species sensitivity dis- CH3OH and H2O in a ratio of 1:2) were used to perform the tribution curve (SSD) and the risk concentrations (HC) of a extractions at 100 °C and 1500 psi in three static cycles, given species can be calculated. For example, HC5, which each withstanding 150 s. The extraction cell was washed means 5% of the species in the species sensitivity dis- with 100% cell volume of fresh solvent. The extracts were tribution curve, shows an effect can be deduced using the diluted with water and processed as water samples to lessen CAFE database and software. The predicted no-effect the concentration of CH3OH (<5 vol %). concentrations (PNEC) can be computed using Eq. 1; Water samples ¼ NOECor EðLÞC50 or HC5PNEC ð1Þ AF The extraction of compounds of interest from water samples The extent of the assessment factor (AF) depends on the was carried out using a solid-phase extraction technique. available data on toxicity. The PNEC values increase Pure methanol (2 × 4 mL) was used to perform the elution. depending on toxicological information for aquatic organ- The eluents were evaporated by a stream of nitrogen and isms obtainable at several feeding levels. Also, the value of restored in their former conditions by adding a mixture of AF decreases in situations of significant and vital datasets. CH3OH and H2O (1:3, v/v). Before analysis, the extracts For example, an AF of 1000 is used when the data on were enhanced with a combination of internal standards to a toxicity is only obtainable depending on E(L)C50, but an final concentration of 15 ng/mL. Each sample was extracted AF of 100 is used when NOEC is obtained from tests with and analyzed three times. one trophic level (e.g., fish). In addition, AF equal to 10 is used when NOEC is known for all three trophic levels Instrumental analysis (Hamre 2006; Molnar et al. 2020). AF is equal to 5 when dealing with five or more different species (separately on High-performance liquid chromatography coupled to mass trophic levels) with the same data on toxicity, which means spectrometry (HPLC-MS/MS) using a technique adapted by the value of HC5 is known. According to Sanderson et al. Gros et al. (2009) was employed to analyze PhACs in the (2004), the predicted E(L)C50 values reported by US-EPA samples. In brief, liquid chromatography was carried out Structure-Activity Relationships Class Program (ECOSAR using SymbiosisTM Pico, stocked with an autosampler, and database) are mainly employed on the condition that no data joined in series to a 4000 QTRAP mass spectrometer on toxicity are available. Although the data on toxicity from stocked with a Turbo Ion Spray source. A Purospher Star the ECOSAR database are immensely in doubt, the accep- RP-18 column (125 mm × 2mm, particle size five μm) was table AF value is 1000 (Zhang et al. 2017). used to achieve chromatographic separation. Eluent A (i.e., a mixture of C2H3N-CH3OH 50%:50% v/v) and eluent B Risk quotient (RQ) (HPLC grade water) were used as the mobile phases for the analysis in negative ionization (NI) mode. The positive Environmental risk assessment can be characterized after ionization (PI) mode analysis was conducted using C2H3N quantifying the PhaACs concentrations in water and surface as eluent A and HPLC grade water with 0.2% formic acid as sediment samples and determining their toxicology thresh- eluent B. The compounds of interest were analyzed in old values. RQ of each PhaACs in each sample was cal- MRM mode, checking two transitions between the most culated using Eq. 2; abundant fragment ions and the precursor ion for the indi- MaximumMEC vidual compound. Detailed information concerning the RQ ¼ ð2Þ operation of the HPLC-MS/MS technique to analyze PNEC PhACs in water and sediment samples can be obtained from Generally, RQ < 0.01 signifies a negligible risk, RQ < other related works [Gros et al. (2009) for water samples; 0.1 indicates a low risk, 0.1 > RQ < 1.0 denotes a med- Jelic et al. (2009) for solid samples]. ium risk, and RQ > 1.0 suggests high ecological risks to Status of pharmaceuticals in the Korle Lagoon and their toxicity to non-target organisms the aquatic organism (EU Commission 2003; Ma et al. sample from sampled site KLW9. Also, ß-blocker atenolol 2016). exhibited a higher average concentration of 86.35 ng/L in a water sample from sampled area KLW8. Toxic unit (TU) Other PhACs detected in almost all water samples include atorvastatin (a cholesterol-lowering statin drug), bezafibrate The degree of a mixture of pharmaceuticals that can harm (lipid regulator), and azithromycin (an antibiotic). Surpris- the non-target organisms can be evaluated by adding the ingly, diazepam, which belongs to the benzodiazepine family concentration (CA) model and ignoring the toxic modes of that acts as an anxiolytic, was detected in all water samples action of the mixture constituents when dealing with a vast and its concentrations were relatively high. Carbamazepine, number of aquatic mixture toxicity studies (Molnar et al. an anticonvulsant medication used primarily to treat epilepsy 2020). The concentration addition model suggests that the and neuropathic pain, was detected in most downstream contribution of the individual toxicants to the total effect sampled sites, but the concentrations were insignificant. can be added in the form of toxicant units (De Zwart, Chloramphenicol drug, which belongs to an antibiotic class of Posthuma (2005)). In this study, the concentration addition medicine for the treatment of several bacterial infections, was of PhaACs was expressed by Eq. 3 (Molnar et al. 2020); detected in most samples collected from upstream and near X MEC the coastal area of the Gulf of Guinea, although the con-n TU ¼ i¼ ð Þ ð3Þ centrations were not high. An antibiotic drug called clari-i 1 E L C50i orNOECi thromycin, which treats strep throat, pneumonia, skin where MECi represents the actual concentration, E(L)C50i infections, Lyme disease, etc., was detected in some water or NOECi represents the exposure concentrations of a given samples collected downstream with significant concentra- PhaACs that can trigger the same toxicity response for all tions. Gemfibrozil drug associated with diet to reduce the compounds. The TU has only one threshold and is a amount of cholesterol and triglycerides in the blood was dimensionless expression, and if its value is >1.0, then a detected in almost all sampled sites; however, its concentra- potential risk is anticipated. tions were insignificant. Ketoprofen which possesses analge- sic and antipyretic effects was observed in samples collected from upstream. However, the concentrations were not high Results (Table 1). Indomethacin, which reduces fever, pain, stiffness, and swelling from inflammation, was detected in some sam- Status of pharmaceuticals in water and surface pled sites; nevertheless, its concentrations were insignificant. sediment samples High levels of mefenamic acid medicine were detected in upstream sampled areas. However, the concentrations detec- Table 1 shows the average concentrations of thirty-five ted in downstream sampled sites were below the limit of pharmaceuticals detected in water and surface sediment quantitation (LOQ). Phenazone and ranitidine were found in samples. Table S1 depicts average concentrations and upstream sampled sites. Comparatively, the levels of raniti- standard deviations (SDs) of PhaACs levels detected (See dine in water samples detected were higher than the levels of supplementary material). phenazone observed in water samples. Tyrosine A was The degree to which the PhaACs in the water samples noticed only in midstream sampled sites; however, the con- are dispersed is below 7% (Table S1). The occurrence tents were insignificant. Tramadol and diazepam were the pattern of each PhACs detected in water samples is similar only PhACs detected in all water samples. No observation of to each PhACs detected in sediment samples with few metronidazole in any of the water samples. exceptions (Table 1). Concerning water samples, the PhACs The dispersion of PhaACs in surface sediment samples is that exhibited a higher average concentration and frequency lower than 8% (Table S1). In sediment samples, there is an of detection include acetaminophen with the maximum observation of higher average concentrations of acet- average concentration of 2987.91 ng/L in a water sample aminophen with 105.11 ng/g in a sediment sample from from sampled site KLW9 and 2622.35 ng/L in a water sampled station KLS1, ibuprofen with 69.21 ng/g in a sedi- sample from sampled area KLW1, Ibuprofen with a max- ment sample from sampled station KLS9, and tramadol with imum average concentration of 105.39 ng/L in a water 56.82 ng/g in a sediment sample from sampled station KLS8. sample from sampled site KLW9. Tramadol with a max- Pharmaceuticals such as bezafibrate, ranitidine, atorvastatin, imum average concentration of 105.11 ng/L in a water ketoprofen, indomethacin, and diazepam were higher in some sample from sampled site KLW6, and Diclofenac with a of the sediment samples (Table 1). Atenolol, clarithromycin, maximum average concentration of 100.91 ng/L in a water fenofibrate, gemfibrozil, josamycin, mefenamic acid, nadolol, sample from sampled area KLW8. Furosemide showed a and tyrosine A were detected at lower levels in all sediment higher average concentration of 157.89 ng/L in a water samples. No detection of metronidazole, trimethoprim, E. A. Asare Table 1 Average concentrations of pharmaceutically active compounds (PhACs) in water and sediment samples from Korle Lagoon, analysis performed in triplicate (ng/L for Conc. in water and ng/g for Conc. in sediment) PhACs Sampled point Water samples KLW1 KLW2 KLW3 KLW4 KLW5 KLW6 KLW7 KLW8 KLW9 KLW10 Anti-diabetics Glibenclamide N.D N.D N.D N.D N.D 14.05 16.27 16.05 9.33 4.91 Psychiatric drugs Diazepam 11.02 9.44 16.10 5.77 5.71 18.81 18.96 10.85 19.07 6.01 Lorazepam N.D N.D N.D N.D N.D