Caffeine as a Pollution Marker for Shallow Groundwaters in a Peri-Urban Area of Campinas / São Paulo – Brazil


 The presence of emerging contaminants, including caffeine, in groundwater can represent an anthropogenic contamination that can impact human health. The present research aims to use caffeine as a wastewater pollution marker of shallow groundwaters in the peri-urban area of Campinas/SP. For a better evaluation of caffeine as an anthropogenic marker, more conventional markers were also analyzed, such as Cl-, NO3-, K+, B, NH4+, NO2- and DOC, and physicochemical parameters such as pH, Eh and EC. Two samplings campaign were done, the first in April (wet season) and the second in August 2019 (dry season), where 18 domestic wells, one sample from the river and one sample from an abandoned meander were selected points to collect water for physicochemical analysis. Nine wells and an abandoned meander waters were selected for the analysis of caffeine. Results show correlations between NO3- and Cl- indicating apport of domestic septic tanks sewage in groundwater. Caffeine, however, was detected in three samples and quantified in one during the wet season and was detected in two samples and quantified in one during the dry season. In the area, caffeine and nitrate have opposite behavior due to reducing conditions of the environment. The study area is rich in dissolved organic carbon, so the conditions for caffeine conservation are not ideal, because it degrades rapidly in groundwaters rich in bacteria and organic matter. Even in this scenario, caffeine was detected in groundwater in the study area, providing unambiguous evidence of anthropogenic pollution of the phreatic aquifer.


Introduction
Over the past few decades, the pharmaceutical personal care products (PPCPs) including the caffeine, have been detected in the aqueous environment worldwide. They are new products or chemicals without regulatory status and whose effects on environment and human health are unknown (Deblonde et al., 2011). PPCPs may enter the aqueous environment directly or indirectly through anthropogenic activities such as sewage discharge, livestock breeding, fertilizing and land ll leachate (Sui et al., 2015).
Shallow aquifers are especially vulnerable to PPCP's because of their high hydraulic conductivity and the lack of con ning layers allowing the in ltration of on-site domestic sewage (Schaider et al., 2014). Caffeine and other PPCP's are unique to human use and provide a relatively unambiguous source identi cation of septic system discharge (Nitka et al., 2019). Caffeine can enter into the domestic wastewater through human urine or household plumbing as it is present at an average amount of approximately 360 mg/L in coffee, tea and soft drinks as a stimulant (Buerge et al., 2003;Sui et al., 2015). In surface waters, caffeine is becoming one of the most used tracers for identi cation of anthropogenic contamination (Buerge et al., 2003). However, concerning groundwater, caffeine can function as a discharge indicator only in certain circumstances where biodegradation is not signi cant, as it degrades rapidly in bacteria-rich groundwater (Knee et al., 2010). Similar to nitrate, chloride, potassium, boron and other inorganic elements, caffeine has been widely used as conventional markers for groundwater contamination in several studies (Huan et 2019) showed that caffeine was detected in groundwater seasonal sampling campaigns and concluded that groundwater was being contaminated by in ltration of wastewater into the aquifer. In contrast, in another study, caffeine was not detected in any groundwater samples presumably due to the microbial degradation in the septic system or within the soil pro le, vadose zone, and/or underlying groundwater (Yang et al., 2017a).
In principle, the bioavailability of organic markers is largely controlled by sorption processes, which are associated with the physical-chemical properties of contaminants, such as molecular structure, water solubility, hydrophobicity and the type of soil (Karnjanapiboonwong et al., 2010;Laws et al., 2011).
The present work intends to use caffeine as an indicator of contamination of shallow groundwater by domestic septic tanks, as well as nitrate, chloride, boron and potassium in the peri-urban area of Campinas / SP, Brazil.

Registration and Sampling
Forty-six hand dug wells were registered in the study area by Alencar (2021). Eighteen of these wells were selected water sampling in two seasonal campaigns -April/2019, rainy season, and in August/2019, the dry season. One sample was also collected from the river and one from the lake of an abandoned meander in each campaign. These samples were tested for the following hydrochemical parameters (pH, Eh, Cl -, NH 4 + , NO 2 -, NO 3 -, K + , B, dissolaved organic carbon -DOC). Nine out of the eighteen wells were selected for caffeine analysis. One sample was also collected from the lake in the abandoned meander. The selection criteria were based on a pre-evaluation of contamination suspicion due to the precariousness of constructive characteristics (domestic well and septic tank), including the conditions around the well and the distance between the wells and septic tanks. The location of the sampling points is presented in Figure 1.
The sampling procedures, including cleaning, ltering, preservation and transport, followed the standards proposed by APHA (2005).
The water samples were collected using disposable bailers, one for each well. Water samples for cations and anion determinations were immediately ltered in the eld using a 0,22 µm polyester membrane lter and placed in polyethylene bottles of 60 ml. Water sample for DOC determination was also ltered in the eld using 0,22 µm polyester membrane lter and placed in amber glass bottles of 30 ml. For caffeine analysis, waters were stored in PET bottles of 1L and vacuum-ltered in vacuum in the laboratory using polyethylene lters with pore size of 0,22 µm and 1L Erlenmeyer ask. All water samples were immediately preserved in the eld after the ltering process in a 4ºC temperature. For cation analysis the samples where acidi ed (1% v/v ultrapure HNO 3 ) for preservation.

Major and Minor Ions Laboratory Analysis
The analysis of cations was performed by mass spectrometry with inductively coupled plasma (ICP-MS) using a device model XSeriesII  (Maria and Moreira, 2007). All the reagents used during the chromatographic procedure were of high purity grade (HPLC grade). For the quality control and to guarantee reliability of the results, the analysis was carried out in triplicates. All the samples were ltered twice using the cellulose ester ltering membrane, rstly with 0,45 μm and secondly with 0,22 μm of pore size (Millipore).
The compound identi cation was done by comparing the retention time of the samples with patterns or with the calibration curve. The quanti cation was done through an external standardization. The pattern curve was constructed with 6 points in which the concentrations varied from 1 to 15 μg/mL of caffeine. The percentage of each analyte in the sample was calculated using equation 1: (1) The groundwater ow net is topography controlled (Figure 2) showing northeast and southeast ows, towards Atibaia river and to lower areas.
Groundwater discharges to the Atibaia river (north area) and to an area with paleo meanders (south area). In this paleo meanders area, the groundwater is owing from east to west area, also discharging in the Atibaia river.

Inorganic Parameters and Caffeine
The results of 11 physical-chemical parameters (pH, Eh, EC, Cl -, NH 4 Potassium (K + ) varied from 0.75 to 16.01 mg/L in the wet season, and from 0.77 to 9.85 mg/L in the dry season. In natural waters, it has a strong tendency to be reincorporated especially in certain clay minerals (Hem, 1985). The Figure 5 (a,b) shows the spatial distribution of ions NO 3 and caffeine. The higher concentrations of nitrate are evident in the west and east regions during the wet season and in the east area in the dry season, Chloride presented similar NO3-behavior.
Caffeine was not quanti ed in samples that had a high concentration of chloride and nitrate. However, caffeine was detected in the west portion area. The concentrations determined in the meander water suggest anthropogenic pollution. Figure 6 (a, b) presents the spatial distributions of DOC and caffeine; K + and B showed similar spatial distribution in wet and dry season, with higher values in the central and south region and lower values in the north area. Similarities in the spatial distribution of these parameters can indicate transport in the same relative speed (Barber et al., 1988). The interaction of caffeine with DOC in the vadose zone can in uence its transport (Yang et al., 2017b). Tables 3 and 4   The positive correlations between B and K + and K + and DOC (Tables 3 and 4, and gures 8a and 8b) can be attributed to the partial dissolution of potassium feldspar and its association with authigenic boron (Rodriguez-Espinosa et al., 2020).

Correlations and bi-variate plots between Inorganic Parameters
EC showed no correlation with DOC in the wet season and positive in the dry season (Table 4 and Figure 8d). The contents of DOC in relation to the meander area, rich in organic matter, are product of the biota living there.

Relationship between caffeine and other parameters.
Caffeine was detected in wells P07, P12, P46 and meander in the wet season, and in wells P07, P46 and meander in the dry season. They are situated in the west portion of the study area and have bad constructive characteristics, precarious septic tanks nearby and poor sanitation conditions in their surroundings. In the abandoned meander water during the dry season, the value of caffeine is higher than in wet season, and the Eh is lower.
It shows that the biodisponibility of caffeine is also widely controlled by sorption processes that are associated with the physicochemical properties of the contaminants, type of soil (Karnjanapiboonwong et al., 2010;Laws et al., 2011), and the volume of sewer discharge. Karnjanapiboonwong et al., (2010) suggests that the adsorption behavior of caffeine is hard to predict simply based in the type of sorbent, considering that caffeine possess a high-water solubility (2.16×10 4 mg/L a 20 o C; low K ow ).
In relation of the four samples in discussion (P07, P12, P46 and Meander), the reducing environment and the presence of organic matter can favor the presence of caffeine in detectable levels (Schaider et al., 2016). On the other hand, this environment is rich in organic matter and bacteria and favors the biodegradation processes of caffeine. It stands out, however, that the tendencies are not conclusive due to the fact that concentrations were bellow analytical quanti cation limit for caffeine.
In all 20 monitored wells in the study area, nitrate showed lower contents in the four wells with detectable levels of caffeine (with the exception of P46, which showed nitrate values of 60.4 and 11.2 mg/L for wet and dry seasons respectively) and relatively higher values in the wells with nondetectable caffeine concentrations. The higher values of NO 3 suggests the septic systems are the main source of NO 3 in groundwaters of the study area (Schaider et al., 2016).
The presence of low levels of caffeine in the groundwaters containing high concentrations of nitrate does not discard the possibility that the residual domestic waters are a source of contamination. The rapid degradation of caffeine in groundwater can justify the absence of this substance in the aquifer. The detection of caffeine can indicate the aquifer recharge by residual domestic waters, even when nitrate is not present in the water (Seiler et al., 1999).
In this study, the low caffeine concentration (above the quanti cation limit) in groundwater and water samples (abandoned meander and domestic wells) can be related to a (bio)degradation processes in presence of DOC concentrations, which is found in higher concentrations. The reduced environment is favorable to the caffeine presence in waters. However, the biodegradation of caffeine is fast in tropical climate alluvial plain, in environments like swamps and marbles, rich in organic material and bacterial ora.

Conclusions
In groundwaters, caffeine can work as a marker of pollution only under certain circumstances where the biodegradation is not signi cant, because it degrades rapidly in groundwaters that are rich in bacteria and organic matter. In the study area, caffeine did not constitute an adequate tracer for contamination studies of groundwater by septic wells because the area is swampy and has high DOC concentrations, an environment where caffeine is likely to undergo rapid degradation.
Despite this fact, caffeine was detected in groundwater and in the lake of an abandoned meander in the alluvial plain of the Atibaia river.
Considering its high speci city to human origin, this is a clear evidence of pollution via domestic sewers wastewater. The reducing environment in the meander and in the swampy region can be the responsible factors for the detection of caffeine in wells with low nitrate concentrations.
In other places in the studied area, Cland NO 3 concentrations indicate pollution in areas with densi cation of septic tanks and poorly built wells.. Potassium and boron were not related to pollution in this case. For the calculation of average and standard deviation of the parameters that presented values below LD, half of the respective value of LD was used. SD=Standard Deviation         The diagrams show the bi variate plots to determine the trend and relationship between a) NO3-vs EC; b) NO3-vs Cl-; c) NO3-vs K+; d) B vs Cl-.