Chemotherapeutic Drugs in Lebanese Surface Waters: Estimation of Population Exposure and Identication of High-Risk Drugs

Environmental risk assessment of anti-cancer drugs and their transformation products is a major concern worldwide due to two main factors: the consumption of chemotherapeutic agents is increasing throughout the years and conventional water treatment processes seem to be ineffective. The aim of the study is to investigate the consumption of anticancer drugs and assess their potential health hazard as contaminants of the Lebanese surface waters. Data on yearly consumption of 259 anti-neoplastic drugs over the years 2013 to 2018 were collected and the following parameters were calculated: yearly consumption of single active ingredients, yearly consumption of drug equivalents (for drugs belonging to the same pharmacologic class/ having the same active ingredient) and Predicted Environmental Concentrations. The classication of compounds into risk categories was based on exposure using Predicted Environmental Concentrations (PECs). The top ve most commonly consumed drugs are Mycophenolate mofetil, Hydroxycarbamide, Capecitibine, Mycophenolic acid and Azathioprine. Based on the calculated PEC values of single active ingredients as well as their equivalents, six high risk priority compounds were identied: Mycophenolate mofetil, Hydroxycarbamide, Capecitibine, Mycophenolic acid and Azathioprine and 5-Fluorouracil. The impact of these micropollutants on animals as well as humans was analyzed. This research paper stresses the importance of further analysis of chemotherapy micropollutants with major focus on high risk drugs. Additionally, regulations should be set in place to ensure proper management of waste water and the development of ecient waste water treatment plants.


Introduction
Cancer is the second leading cause of death in the world. Every sixth death in the world is due to cancer (1). In Lebanon, the number of new cancer cases diagnosed in 2018 is 17294 cases (288 cases per 100,000 population) with breast, bladder, lung, prostate, and colorectal cancers being the most common (2). The number of death from cancer has doubled between the years 2000 (3776 deaths) and 2017 (6639 deaths) with a relative increase in less than 1.5 M in Lebanese population (2). As the world population is growing and aging, the global number of cancer death is expected to increase further. Chemotherapy is often used as a major treatment for almost all types of cancer.
Cytotoxic drugs are a group of compounds used in chemotherapy to prevent or disrupt cell division. There are over 50 cytotoxic drugs used routinely in chemotherapy. Chemotherapeutic agents can be classi ed into different categories based on their chemical structures and the way they act on cancer cells. Most commonly used agents are alkylating agents, which act directly on DNA, causing cross-linking of DNA strands, abnormal base pairing, or DNA strand breaks, thus preventing the cell from dividing. Another class of commonly used chemotherapeutic drugs are antimetabolites, which replace natural substances as building blocks in DNA molecules, thereby altering the function of enzymes required for cell metabolism and protein synthesis. Plant alkaloids are antitumor agents derived from plants. These drugs act speci cally by blocking the ability of a cancer cell to divide and become two cells. Another major class are antitumor antibiotic. They act by binding with DNA and preventing RNA synthesis, a key step in the creation of proteins, which are necessary for cell survival. Many of these drugs are mutagenic, carcinogenic, teratogenic and/or toxic to reproductive systems and are classi ed as highly hazardous compounds (3).
Anticancer drugs present as a major water contaminant worldwide; Lebanon's case is not less worrisome. Environmental risk assessment of anti-cancer drugs and their transformation products is a major concern worldwide; studies have been conducted to analyze chemotherapy drugs consumption trends, availability in surface water and population exposure. This is due to two main factors: the consumption of chemotherapeutic agents is increasing throughout the years and conventional water treatment processes seem to be ineffective. Many research studies reported that wastewater treatment plants, even those that meet regulatory requirements for waste water treatment, are only moderately effective in removing the aforementioned pharmaceuticals[(4-7). The pharmacologic properties and mutagenic properties of chemotherapy drugs potentially make anticancer drugs the most dangerous contaminant of our water system and pose a risk to the aquatic life and human beings. Available ecotoxicological data for anti-cancer drugs and their metabolites are incomplete. So far, the situation in Lebanon was not evaluated by any environmental risk assessment studies. Our work is the rst to present an overview of the consumption trends in Lebanon, to forecast the presence of the associated contaminants in surface waters, and to conclude with an assessment of the correlated risks. The assessment will take into consideration the pharmacokinetic properties (metabolism and excretion) of each of the anticancer drugs analyzed.
The aim of the study is to investigate the consumption of anticancer drugs and assess their potential health hazard toxic effects as contaminants in the environment.

Materials And Methods
The work is divided into ve steps: Study the consumption of chemotherapy drugs in Lebanon over the years 2013 to 2018.
Compute the concentration of these drugs in surface water.
Investigate the exposure of the Lebanese population to anticancer drugs.
Assess the long term risks associated with this exposure and the potential effect on Lebanon as well as countries on the Mediterranean Sea.
The brand names of cancer drugs available on the Lebanese market and registered at the Ministry of Public Health (MOPH) were identi ed, and the yearly consumption of each drug consumed between 2013 and 2018 was studied. Data comprised all the dosage forms available on the Lebanese market including tablets, capsules and injectable drugs. As per the MOPH, anticancer drugs are classi ed as: endocrine therapy, immunosuppressive agents and antineoplastic agents.
1.2 Data collection and concentration in surface water.
Consumption data was identi ed for 259 anti-neoplastic drugs over six years. The total amount of active ingredients consumed over the years was calculated based on the following: For every brand drug, consumption per year (in mg) was calculated using the number of boxes (preparations) consumed x the number of units/ box and the amount of active ingredients in each unit (in mg).
Drugs with the same active ingredient were grouped together and their consumption was summed up.
When applicable, different active ingredients belonging to the same pharmacologic or chemical class were grouped together. Total consumption in every drug class was calculated using conversion factors.
Cumulative concentration was calculated by summing the total consumption per year 2013 to 2018 as provided in Table 1. Pharmaceuticals that are used in higher amounts have a potential to reach the aquatic environment in greater quantities and therefore present a higher risk to human and aquatic life. Therefore, risk classi cation is done according to quantity consumed. The classi cation of compounds based on exposure is done using Predicted Environmental Concentrations (PECs), according to Scheme 1. Thresholds for risk classi cation adopted in the analysis are 100 ng.L -1 (guidance by the Food and Drug Administration-FDA (14) and 10 ng L -1 (guidance by European Medicine Agency-EMEA (15) Two PEC values were determined: PEC a assumes no metabolism of the active ingredient happens in the body, 100% of the active ingredient is excreted unchanged.
Where Consumption is the amount of active ingredient consumed per year (ng year -1 ) WWinhab is the water consumption per person per year, Inhab is the number of inhabitants of Lebanon and DF is the dilution factor from waste water treatment plants e uents to surface water.
PEC b is calculated when metabolism data is available for the drug. The fraction of active ingredient excreted unchanged (F exc ) is added to the equation.
PEC c is the Predicted Environmental Concentration in surface water that remains after treatment. Therefore, PEC c is obtained by taking into consideration the fraction of pharmaceuticals removed by waste water treatment plants (F WWTP ). However, due to the absence of e cient treatment plants or the absence of data in Lebanon, the F WWTP was assumed to be equal to zero. PEC c and PEC b values are therefore equal.
The following assumptions were made when using the formula: According to the EMEA guidelines, the amount of wastewater per inhabitant per day to be equal to 200 L. The yearly consumption (WWinhab) would therefore be equal to 7300 L inhab -1 year -1 (15) The number of inhabitants in Lebanon was assumed to be equal to 6,000,000 (16).
The Dilution Factor was assumed to be equal to 10 as per the EMEA guidelines.
The fraction of active ingredient excreted unchanged was assumed to be equal to 0.5 when data was not available.

Toxicity classi cation
Depending on PEC a and PEC b values obtained for each drug, a different class was assigned to each drug. Drugs were categorized into six classes from the highest to the lowest risk, as explained in Table 2 and scheme 1.  The most consumed drug, mycophenolate mofetil, also known as MMF, is an ester prodrug of mycophenolic acid (MPA), classi ed as a reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH). Once orally administered, MMF is rapidly converted into its active form MPA by hepatic carboxylesterases 1 and 2 (18,19). Therefore, MMF should be considered as a source of MPA in terms of environmental contamination. Accordingly, consumption data of both drugs were grouped together based on the conversion factor presented in table 1. The yearly consumption of both drugs and their equivalent total MPA is presented in gure 2.
The second most commonly consumed drug is Hydroxycarbamide, also known as hydroxyurea. Hydroxycarbamide is an antineoplastic agent that inhibits DNA synthesis through the inhibition of ribonucleoside diphosphate reductase. Once administered, Hydroxyurea is converted to a free radical nitroxide (NO) in vivo (20). The third most commonly consumed drug is capecitabine, an orally-administered chemotherapeutic agent used in the treatment of metastatic breast and colorectal cancers. Capecitabine is a prodrug that is enzymatically converted to uorouracil (antimetabolite) in the tumor, where it inhibits DNA synthesis and slows growth of tumor tissue (9,21). The environmental toxicity of capecitabine should therefore be analyzed as part of that of Fluorouracil. The yearly consumption of both drugs and their equivalent 5-FU is presented in gure 3. The forth most commonly consumed drug is mycophenolic acid and the fth is azathioprine, a purine analogue with cytotoxic and immunosuppressive activity. It is prodrug of 6-mercaptopurine (22). Figure 4 illustrates the change in the consumption of each of the commonly consumed chemotherapeutic drugs over the years 2013-2018.
In the rst section, the results illustrate the amount of anticancer drugs consumed in Lebanon. In this section, however, in order to better re ect on the exposure to anticancer drugs, several factors are considered (excretion and waste water treatment plants removal rates). First, the concentration of each anticancer drug in surface waters was predicted without regard to transformation in the body and removal at WWTPs. This value (PEC a, ) is an illustration of the worst case scenario. Potentially hazardous compounds, belonging to class II, include azathioprine, capecitabine, Hydroxycarbamide and Mycophenolic acid, and 5-uorouracil equivalent. Therefore, from the 259 anti-neoplastic agents available on the Lebanese market, the previously enumerated 6 compounds should be considered as priority for further evaluation. Figure 5 presents the PEC b value for the priority anticancer drugs. Table 3. Risk categorization of chemotherapeutic drugs based on environmental exposure data between 2013 and 2018.  MMF is the most commonly consumed drug. The main reason behind the elevated consumption of MMF prodrug and its active ingredient MPA is that they are used for as both chemotherapeutic and immunosuppressive agents in organ transplantation as well as auto-immune disease (19). The analysis of the toxicity of MMF and MPA each separately led to an underestimation of the risks imposed by this drug. Since MMF is a prodrug of MPA, the consumption of both was grouped using drug-speci c equivalence factors under a total MPA equivalent (  (17). As for mycophenolic acid, its consumption in Lebanon equals 72.03287 μg Inhabitant -1 Day -1 , higher than in Portugal (50 μg Inhabitant -1 Day -1 ) ) (17) and Poland (51 μg Inhabitant -1 Day -1 ) (23)but lower than in Catalonia (704 μg Inhabitant -1 Day -1 ) (24).
The second most commonly consumed drug is hydroxycarbamide. This could be explained by the fact that hydroxycarbamide is highly consumed in the Middle East and Africa region and it is relatively cheaper than other chemotherapeutic drugs and has several indications (25 (24) and France (284 μg Inhabitant -1 Day -1 ) (27).
The third most commonly consumed drug is Capecitibine. Capecitabine is a widely used chemotherapeutic drug as it has gained approval in two of the most common cancers in Lebanon: breast and colorectal cancer (2). Additionally, There is a global trend towards the prescribing of capecitabine over uorouracil as capecitabine allows more patient-friendly oral administration, improved quality of life and fewer side effects (28). It is worth noting that consumption data of uorouracil for the years 2014 to 2016 was absent from the provided data. Accordingly, we could not have a clear image on the extent/trend in its consumption. As previously mentioned in the results section, capecitabine is a prodrug of 5-uorouracil. Accordingly, the consumption of both was grouped using drug-speci c equivalence factors under a total 5-uorouracil equivalent. The PEC values calculated for the equivalent lead to its risk categorization into class IIA for the years 2013-3018, indicating that 5-uorouracil is a potentially hazardous compound. This again proves that a separate analysis of capecitabine In this study, we have analyzed the risk imposed by chemotherapeutic micro-pollutants based on their yearly consumption. However, in fact, some chemotherapeutic drugs accumulate and their amounts in the environment build up over the years, alluding to a higher ecotoxicological risk. A review of the environmental fate of the top 5 most consumed drugs reveals that none of them is a potential bioaccumulative drug (9,28,30). However, less commonly consumed chemotherapy drugs that can accumulate and potentially pose a serious environmental risk include: Cyclophosphamide, Ifosfamide (31) and Erlotinib (32).
Another factor that should be taken into consideration is that the residues of pharmaceuticals occur in the environment as complex mixtures, and therefore, even though the concentrations of an individual compound might be low, the cocktail effect might have a more concerning ecotoxicological implication. Research studies on the environmental concern of combinations of anticancer drugs revealed that the interactions of anticancer drugs could be of environmental concern (33-35).

Impact on animals/humans.
Studies investigating the toxicity of chemotherapy drugs on animals are scarce and are usually done using higher concentrations than those found in the environment. Accordingly, additional work is needed to evaluate the acute and chronic risks associated with exposure to whole-transcriptome changes (37). Another study by  reports an increased frequency of abortive grains in higher plants exposed to 5-uorouracil (38 Pharmaceutical compounds are designed to have desirable target effects in the human body, but the impact of chronic indirect exposure to these compounds can be dangerous for both humans as well as other species. Anticancer drugs have potent cytotoxic, genotoxic, mutagenic, carcinogenic, endocrine disruptor as well as teratogenic effects. The augmentation of cancer disease in the Lebanese population has led to an increase in the drug consumption and therefore an increase in drug discharge into the environment. Therefore, it is indeed essential to pay special attention to the discharge of anticancer drugs in the environment and the development of effective waste water treatment plants. It is worth noting that not all waste water treatment techniques are effective at removing anticancer drugs. The scarce literature available shows that a promising method for the removal of micropollutants is the membrane bioreactor (MBR), which combines physical removal with biological removal (biodegradation) to improve the outcome of the water treatment (6).

Pharmaceutical waste management in Lebanon.
In this paper, we have tackled the risks imposed by a major source of chemotherapeutic drug in surface waters: the chemotherapeutic drugs consumed by the patients in medical centers as well as their home. However, another major source of micropolluants are expired/unused drugs that stay at the importer's warehouses and do not reach hospitals/patients. In the absence of a safe solution and the necessary regulations to dispose of pharmaceutical surplus, warehouses in Lebanon are lling up with old, expired drugs (39).

Need for regulations
Efforts are needed to set new policies regarding the disposal of micropollutants in general, and chemotherapeutic drugs primarily. We hope that this research study sheds light on the risk imposed by chemotherapeutic drugs and contributes to future establishment and enforcement of regulations to protect the public from the danger of such micropullutants in surface waters.

Future studies needed
Our research study is the rst of its kind in Lebanon and the Middle East. As previously stated, in this study, we have estimated the environmental concentrations in surface waters by the calculation of predicted environmental concentrations based on consumption data. Reports on the occurrence of chemotherapeutic drugs in surface water, ground water or drinking water by measurement of actual of the drug concentrations are absent.

Conclusions
This work presents, for the rst time, an extensive analysis of the consumption rates of 259 different anticancer drugs dispensed in Lebanon between 2013 and 2018. Globally, there is a growing attention regarding the fate of pharmaceuticals in the environment and their potential toxicity to different species. All pharmaceuticals, by design, are meant to elicit a biological response. Chemotherapeutic drugs are of a particular concern, as these drugs were designed to damage DNA, inhibit DNA synthesis, and interrupt cell replication. They act unselectively on all growing cells and have mutagenic potency. The analysis showed that the top ve most commonly consumed drugs are: mycophenolate mofetil, hydroxycarbamide, capecitabine, mycophenolic acid and azathioprine. A preliminary analysis of the toxicity risk based on the assessment of exposure allowed the identi cation of six potentially priority drugs, belonging to classes IA (high risk compounds) and IIA (potentially hazardous compounds): mycophenolate mofetil, hydroxycarbamide, capecitabine, mycophenolic acid, azathioprine and 5-uorouracil. Our analysis has also proven that, when analyzing the potential risk of such micropollutants, it is a key prerequisite to combine drugs with common end product, as their toxicity cannot be separated. Further analysis of these priority micropollutants is necessary, with actual measurement, rather than estimation, of their occurrence in surface waters. In this study, we have shed light on potential contaminants of our surface waters, emphasizing the importance of e cacious waste water treatment plants and the enforcement of regulations with regard to environmental pollution. Declarations