Determination of Caffeine, a specific marker for wastewater contamination, in coastal
environments from the Florida Keys
Piero R. Gardinali*1,2, Arlette Azua 1 and Joseph Boyer 2
1Department of Chemistry, and 2Southeast Environmental Research Center (SERC),
Florida International University, University Park. Miami, FL 33199
Recently, there has been a growing interest in the presence of pharmaceuticals in the
aquatic environment. Pharmaceuticals are developed with the intention to perform a
biological effect so there is no reason to believe that they will be “environmentally
harmless”. Generally, drugs are absorbed by the organism and subject to metabolic
reactions. However, a significant amount could leave the organism unchanged via
urine or feces and enter sewage or manure. In addition, recent studies show that
elimination of pharmaceuticals in municipal sewage treatment plants (STP) is often
incomplete. The Florida Keys have approximately 85,000 permanent residents and
2.5 million tourists each year. Most of the Keys do not have modern sewagetreatment plants and its population is mainly served by septic tanks, underground
injection wells and as much as 10,000 illegal cesspits. This fact, combined to the
existence of waterfront properties sitting on a porous limestone foundation creates a
good opportunity for water quality degradation via wastewater intrusion. To assess
the infiltration of human derived waste and to check for the presence of
pharmaceuticals coastal waters, caffeine was selected as a probe mainly because of its
anthropogenic nature, the uniqueness of its origin, its environmental fate, and its
elevated consumption. An average person consumes and discards large amounts of
caffeine daily. Depending on its origin, freshly brewed coffee contains between 10 to
400 mg/L (parts per million) of caffeine while caffeinated sodas have between 100
and 130 mg/L consequently a single person can generate hundreds to thousands of mg
of caffeine per day. The liver extensively metabolizes caffeine and only about 3%
leaves the body unmetabolized. Thus, the major source of caffeine in domestic
wastewater comes from unconsumed coffee, tea, soft drinks, or medication moving
through ineffective on-site wastewater treatment systems (septic tanks). Crowded
areas of septic systems present a high risk of microbial fecal contamination to
groundwater and general degradation of water quality. Studies in the Florida Keys
have already shown widespread bacterial and viral contamination of surface waters
often associated with septic systems. Our study area concentrates in the Florida Keys,
mainly the Little Venice area (Marathon Key) but also includes several other canal
systems from Key Largo to Key West serviced by both septic tanks and wastewater
treatment plants.
Caffeine was either extracted by LLE or SPE. In liquid-liquid extraction, a one-liter
surface water sample is fortified with 50 μl of 1.000 ng/ml of atrazine-d5, used as the
internal standard. Then, 20 g of NaCl are added to increase the ionic strength of the
solution followed by 1.00 ml of 1M K2PO4 buffer to bring the water to pH~8.1. After
each extraction with 50 ml of MeCl2 the organic layers are passed through anhydrous
sodium sulfate and concentrated in a water bath to about 1 ml. After taking the sample
to dryness using a flow of clean nitrogen, the samples are reconstituted with 500μl
50:50 MeOH:H2O and analyzed using LC-APCI+-MS.
In the solid-phase extraction method, a 500 mg Oasis® HLB plus cartridge was used.
The cartridge was conditioned with 5ml MeOH followed by 5ml H2O. A 1 L water
sample containing 20 g of NaCl at pH~8.1 and 100 μl of internal standard was filtered
and passed through the cartridge using a multichannel peristaltic pump at a rate of 10
ml/minute. Elution of caffeine and atrazine-d5 from the cartridge was achieved with
2 ml of a solution of 1% Acetic acid in 80:20 MeOH:H2O followed by 2 ml of
methanol. The combined eluents were then evaporated to 500 μl and analyzed using a
Finnigan navigator aQa LC-MS operating under positive ion atmospheric pressure
chemical ionization (APCI+) in selected ion monitoring (SIM) for the [M+H]+
pseudo-molecular ions. The corona pin voltage was set at 4.5kv, the fragmentation
cone voltage at 15V, and the source temperature at 350°C. For the HPLC separation,
a Luna C-18 reverse phase column (150 x 4.6 mm, 5) was used with a linear
gradient from 30:70 MeOH/H2O to 100% MeOH for 10 min. The flow rate was set
at 1.0 ml/min. The ions analyzed in the SIM mode for caffeine were 195 and 196 and
for Atrazine-d5 were 221 and 223Caffeine was detected in a large number of the
samples collected ranging from the Miami River to Key West Municipal Marina.
Concentrations of caffeine in open waters are generally low and near the method
detection limit (<4 ng/L) while areas affected by water quality problems had
concentrations of caffeine between 30 to 200 ng/L respectively. Specifically for Little
Venice, occurrence of caffeine was widespread and more frequent in canals and
enclosed waterways (LV01 to 09) than in open bay waters (LV-10). No significant
differences in concentrations were observed between the mouth and head of the
canals but the station located at the end of the 112th street canal (LV-03) was
consistently higher than other stations in the survey. Although correlations with other
water quality parameters such as nutrients (nitrates, nitrites, total phosphorous,
reactive phosphorous, TOC, chlorophyll, etc.) and bacterial counts (FC and EC) were
generally poor (r2 < 0.2) station LV-03 still had the highest levels for most of these
parameters during the period sampled. No trends were also apparent between the
caffeine concentrations and average rainfall. These results point out the complexity of
water circulation in the canal systems where a large tidal exchange between Florida
Bay and the Atlantic Ocean waters tend to dominate the saltwater circulation.
Preliminary data on water stratification indicates that caffeine concentrations are
elevated in well-mixed systems when compared with stratified ones. To validate this
observation, collection of both surface and bottom waters is already underway.
The relevance of these findings both simple and profound; if caffeine is present in the
water, many other “biologically active” pharmaceuticals and personal care products
could be present as well. Future research is needed to establish not only the
occurrence but also the extent of this wastewater contamination and the potential
effects
to
near
coastal
ecosystems
at
risk.