The Effect of Soil Type on Migration of Pharmaceuticals and

Personal Care Products (PPCPs) Through Cape Cod Soils Semester in Environmental Science

December 21, 2015 Theodosia Fehsenfeld

Colorado College Advisors: Dr. Maureen Conte and JC Weber

A. Abstract Thirty to ninety percent of Pharmaceuticals and Personal Care Products (PPCPs)

do not degrade after ingestion into the body (Bio Intelligence Service 2013). This means that they remain bioactive when they enter the wastewater system and ultimately the environment. Compounds not removed in wastewater treatment have the potential to contaminate underground aquifers. Retention in soils depends upon their chemical structure and surrounding soil type. The octanol water coefficient (Kow) is the relative compound’s affinity for water, a polar solvent and n-octanol, a non-polar solvent (Leo et al. 1971). The Kow is a good indicator of the relative water solubility of various compounds (Smith et al. 1988). Therefore, compounds with a low Kow are more likely to enter groundwater resources; where as, compounds with high Kow are more likely to be retained in soils. Soil type also affects the rate of transport of pharmaceuticals through soils. The presence of Soil Organic Matter (SOM) has been found to retard PPCP migration through soils (Gibson et al. 2010). Cape Cod soils are primarily sandy and therefore lack organic matter that slows PPCP migration, increasing the risk of infiltration of PPCPs into groundwater, threat to marine and terrestrial biota, and human health. This study analyzes rates of migration of PPCPs through sand and organic-rich soils of the West Falmouth region of Cape Cod. In a controlled experiment, groundwater flowed through cores containing both soil types at the average groundwater flow rate observed on Cape Cod (15-30 cm/day) (Standley et al. 2008). A spike of six different PPCPs commonly used on Cape Cod (clofibric acid, ibuprofen, 6-Acetyl-1,1,2,4,4,7-hexamethyltetraline (AHTN), 4-Methyl-benzylidene (4MB), triclosan and β-estradiol (Kow 0.16-5.92 range)) were added to the tops of the cores. Migration was tracked throughout a period of six days. The presence and concentrations of PPCPs were analyzed using GC-MS techniques. Hydrophilic compounds, such as clofibric acid and ibuprofen which have a lower Kow, traveled at a faster migration rate and passed through the cores within 24 hours. Hydrophobic compounds (higher Kow) such as triclosan, AHTN and 4MB were mostly retained in the cores and had a slower migration rate. β-estradiol which had a medium Kow value when compaed to the other compounds investigated, partitioned throughout both the soil and groundwater. Detectable concentrations of estradiol, triclosan and 4MB (3.5, 6.5, and 4.4 ng/L) were found in the groundwater used in the experiment at levels one order of magnitude higher than values found by Zhang et al. (2014) and Standley et al. (2008) studies, which were conducted on Cape Cod, indicating contamination from either proximal household septic systems or the Falmouth Wastewater Treatment facility located 2 km upstream. Key phrases or words Octanol water constant (Kow), Pharmaceuticals and Personal Care Products (PPCPs), hydrophilic, hydrophobic, soil matrix, soil organic matter, solid phase extraction (SPE), GC-MS

B. Introduction

Hundreds of tons of PPCPs are produced and consumed annually in the developed

world (Karnjanapiboonwong et al. 2010). However, PPCP presence in the environment

has only been intensively studied in the last ten years (Ibid.). The main sources of PPCP

runoff are wastewater and agricultural runoff (Ibid.). Use of recycled water has become

more common especially in the western United States due to water conservation efforts,

increasing the likelihood of pharmaceuticals to re-enter food supplies and ecosystems

(Chefetz et al. 2010).

Large amounts of some pharmaceuticals are excreted from the body fully intact or

slightly altered. Many chemicals that are conjugated in the body have the ability to re-

form into their previous conformation in wastewater (Karnjanapiboonwong et al. 2010),

resulting in many organic compounds considered as “bioactive” in wastewater effluents

due to little or no degradation. (Karnjanapiboonwong et al. 2010). The prevalence of

these organic compounds in effluents has implications for contamination of groundwater

aquifers and nearby surface waters.

On Cape Cod, soils are sandy and have low percent organic carbon resulting in a

greater porosity, increasing the likelihood of pharmaceuticals to leach into groundwater

(Standley et al. 2008). Zhang et al. (2014) found detectable presence of pharmaceuticals

such as clofibric acid and estrogen in groundwater surrounding estuaries near wastewater

effluents in Falmouth, MA.

Understanding how these organic compounds move through the soil is crucial to

providing appropriate measures for remediation and reduction of contamination. While

not all PPCPs persist in the environment over long periods of time, they are used

frequently and accumulate overtime, and therefore, we must understand their migration

through different soil types (Karnjanapiboonwong et al. 2010).

Past literature identifies octanol water constants (Kow), hydrophobicity, polarity,

solubility and the presence of SOM and Dissolved Organic Matter (DOM) as important

characteristics influencing the rate at which organic compounds move through soils

(Standley et al. 2008). In semi-arid climates where SOM and clay are low,

pharmaceuticals have a higher potential to migrate into groundwater sources (Chefetz et

al. 2010, Gibson et al. 2010). Kulshrestha et al. (2004) speculated that monovalent

cations on SOM increased the number of binding sites initiating retardation of organic

compounds. Gibson et al. 2010 found that hydrophobic chemicals tend to be retained in

sludge, while hydrophilic compounds are released with wastewater effluent. Therefore,

more soluble chemicals have a higher potential to seep into groundwater sources,

whereas hydrophobic compounds may be retarded by upper soil layers and be taken up

by plants or affect microbial communities (Ibid.). Other factors such as soil pH,

temperature and photo radiation also have major effects on sorption of pharmaceuticals to

soils but they are not considered in this study.

We conducted a controlled experiment to compare migration of a suite of organic

chemicals with varying hydrophobicity (clofibric acid, ibuprofen, 6-Acetyl-1,1,2,4,4,7-

hexamethyltetraline (AHTN), 4-Methyl-benzylidene (4MB), triclosan and β-estradiol)

through two soils types: sandy soil with low SOM (typically what is found on Cape Cod)

and more organic carbon rich soil.

Based on previous literature and the molecular characteristics of each compound,

we hypothesize that more hydrophobic compounds will be retarded more readily in the

soil, whereas hydrophilic molecules will be more soluble in the groundwater and travel

more rapidly through the soils. Pharmaceuticals will travel less readily through soils rich

in organic matter because of interactions with monovalent cations on SOM and increased

numbers of binding sites (Kulshrestha et al. 2004).

C. Methods

Target Compound Selection

Seven PPCP compounds were selected because of their varying chemical properties and

their common use by residents of Cape Cod and the United States (Table 1).

Sample Site Selection

A mudflat 200 m up the Mashapaquit Creek was chosen as a study site because of the

presence of an established well and its easy accessibility (Fig. 1). Also, this site contained

observable differences in soil types suitable for the experiment. Coincidentally, this site

is impacted by a groundwater plume that originates from the Falmouth Wastewater

Treatment Plant, 2 km upstream.

Collecting Soil and Water

Samples were collected at a private residence on the Mashapaquit Creek, the only

freshwater inlet into West Falmouth Harbor. Ten gallons of groundwater was collected

from 20 m below ground from an established well and initially checked to confirm that it

was from a freshwater source using a refractometer. The water was filtered using GF/D

filters in a Swinex filter cartridge and retrieved using a Geotech Geopump Peristaltic DC

Pump. Soil was collected from two adjacent patches, 0.25 m deep, and transferred to 5

gallon buckets. The soil with organic matter was dark grey in color, had a much smaller

grain size than the sandy soil and held more water while the sandy soil was tan in color

with larger grain sizes. Both types of soils were sieved using 2 mm sieves, homogenized

and stored at 15 ° C with the groundwater for four days until the beginning of the

experiment.

Characterization of Soils

Both soil types were analyzed to determine quantitative differences between soil

moisture, percent organic carbon, total carbon and nitrogen, and SOM presence. For soil

moisture, the soils were weighed before and after drying at 60 °C for two days. For C and

N analysis, soils were initially dried at 60 °C for two days, and ground with a mortar and

pestle. Samples were than weighed out according to their expected carbon percentage (ie.

Sandy soil ~ 10-15 g and organic rich soil ~ 3-5 g) and combusted using a 2400 CHN

Elemental Analyzer. For organic carbon, samples were initially incubated and ground

finely using the same procedure for total carbon but they were also acidified by adding

150 µL of 4% sulfurous acid to remove carbonates that may have been present in the

sample. For SOM presence, dried samples were combusted at 465 °C for four hours in a

muffle furnace and the difference in mass before and after combustion was determined as

the percent SOM.

Obtaining the Correct Flow Rate

To obtain a constant groundwater flow rate of 25 mL/day manually, a Mariotte bottle was

constructed by drilling a 3 cm diameter hole in the top of a ten-gallon carboy to fit a

rubber stopper with a glass rod placed through its center that was long enough to reach

the bottom of the carboy (~ 35 cm). The carboy was filled with collected groundwater

and 12 0.38 mm diameter polyethylene tubes that were two meters in length were

threaded through the glass tube until they reached the bottom of the carboy. Each tube

was threaded through a 27-guage needle that pierced a rubber septa that enclosed the top

of the core. Twelve 35 cm long teflon tubes were mounted vertically and filled with

either soil type leaving 5 cm of space on each end. A Teflon frit and 3 cm of sand was

placed into the tube prior to adding the soil type to reduce the potential of clogging.

Each tube was marked to create three equal sections and slid into a black plastic tube to

simulate a lack of light exposure.

Applying PPCP Spike

The soil columns were preconditioned with groundwater for one day before addition of

the spike. After preconditioning, the water level in each core was lowered to the top of

the sediment and approximately 20 µg of each of the six PPCPs was added to each core

by pipetting 0.1 mL of a PPCP standard mix which contained 244.43 µg/mL of clofibric

acid, 202.22 µg/mL of ibuprofen, 234.22 µg/mL of estradiol, 281.88 µg/mL of triclosan,

241.71 µg/mL of AHTN and 233.54 µg/mL of 4 MB. Flow was then resumed to

approximately 25 cm/day groundwater rate.

Collecting Soil, Water and Recovery Samples

During the course of the six-day experiment, water from each core was collected in clean,

pre-weighed glass vials roughly every 12 hours depending on flow rate. Flow rates were

calculated for cores analyzed (Core 3 and 6) according to the measured volume of

effluent (Fig. 5 and 6). A sediment core of each soil type was harvested every 24 hours

for six days. Each core was divided into three equal sections, transferred to pre-weighed

Teflon tubes and frozen until analysis. Three aliquots of each soil type were spiked with

the same ~ 20 µg of each PPCP added to the cores, and frozen to analyze for recovery

efficiency.

Isolation of PPCP

The extraction process was modified from Gibson et al. 2010.

Sediment samples were freeze-dried using a Virtis freeze dryer.

10 µg of the internal standard, 21:0 178 µg/mL fatty alcohol was added to each dry

sediment aliquot before PPCP extraction. To extract the PPCPs, 30 mL of 1:1

acetone:ethyl acetate reagent was added to each sediment tube. The tubes were shaken

vigorously and ultra-sonicated for 15 minutes. The samples were kept from overheating

by immersing in a recirculating ethylene glycol bath cooled with dry ice to 15-20 °C. The

tubes were centrifuged for 10 minutes at 3000 rpm to fully separate the sediment and

solvent. Then the supernatant was removed from the sediment tubes and transferred to

corresponding pear flasks. This extraction was repeated a second time, combining the

rinses in the same pear flask. The solvent was evaporated to just dryness using a

rotoevaporator and resuspended in 0.5 mL1:1 acetone:ethyl acetate and diluted with 50

mL of Milli-Q + water. For the extraction blank, we added PPCP mix (~ 20 µg each) to a

pear flask and diluted with 50 mL of Milli-Q+ water. Between 3-5 drops 12M HCl was

added to each pear flask to reduce the extract to pH 2 before loading it onto the SPE

cartridge.

The PPCPs were isolated using ThermoFisher Hypersep C18 cartridges (500 mg/3

mL bed). Cartridges were preconditioned with 5 mL of Milli-Q + water, 10 mL methanol

and another 10 mL of Milli-Q+ water. Samples were loaded onto SPE cartridges at a rate

of 3 mL/min controlled with positive pressure from zero-grade nitrogen gas. After all

samples were loaded, SPE cartridges were dried with nitrogen gas for one min.

PPCPs were eluded using solvents with ranging polarities. To elute acidic compounds, 5

mL of 40:60 acetone : 0.1 M Na bicarbonate solvent was added to each SPE and eluded

at 3 mL/min. The extract was collected in a clean test tube. To elute nonacidic

compounds, 6 mL of acetone was passed through the SPE columns and combusted

NaSO4 columns to remove residual water. This second elution was collected in an

additional set of 13 mL test tubes. The tubes with the nonacidic compounds were

evaporated to just dryness using the Savant Speedvac sc110 and re-suspended in 0.5 mL

acetone. The acidic fraction was extracted with ethyl acetate (2 times 2 mL), passed

through NaSO4 columns and combined with the nonacidic fraction. Samples were

evaporated to dryness in the Speedvac and resuspended in ~ 50 µL methylene chloride

(DCM).

Derivitization and Analysis by the GC/MS

TMS-Derivitization was used to prepare compounds for analysis by the GC/MS by

replacing hydroxyl groups on the compounds with trimethylsiyl groups. The extract

suspended in DCM was transferred to GC/MS vials followed by ~ 50 µL of DCM to rinse

the test tube and transfer as much sample as possible. Using a fixed volume Drummond

pipet, 25 µL of pyridine and 25 µL of BSTFA + 1% TMS was added to each vial. Vials

were incubated at 55°C for 1 hour. Samples were then dried with N2 and resuspended in

100 µL of DCM for GC/MS analysis.

Running the samples on the GC/MS

Samples were analyzed using an Agilent GC/MS with a CP-Sil SCB column

(60mx0.25mmdia x 0.25µm film thickness) at 50 °C (5 minute hold) ramping at 5

°C/minute to 320 °C with a 20-minute hold.

Quantification

Using Agilent Chemstation software, the area (abundance) of each PPCP target ion was

retrieved and using the PPCP calibration, each PPCP compound in the sample was

quantified (Table 2 and Fig. 4). Samples were corrected for recovery efficiency (Table 5).

PPCP Standard Calibration

Five GC-MS vials were spiked with 10, 8, 6, 4 and 2 µg of each PPCP from the standard

mix (including one replicate of the 8 and 4 µg standards) and resuspended in 100 µL

DCM. They were derivitized using the same reagents and protocol as the samples. GC-

MS injections of 1 and 2 µL were used resulting in calibration ranges of 20 – 200 ng.

Calibration curves were established for each PPCP compound using the area of the target

ion for each respective compound relative to the amount of PPCP injected (Table 4, Fig.

4). The target ion is one particular ion from the mass spectra used to quantify the area of

the peak to find the abundance of the compound in each sample. A calibration was also

established for a second target ion to ensure no inconsistencies. The calibration curves

with the primary target ion were used to quantify PPCPs in the samples (Fig. 4). All the r2

values were between 0.89 and 0.97 for the calibration curves. This indicates a linear

correlation between the amount of PPCP (ng) and the abundance based on ion area (m/z)

(Table 4).

D. Results

A series of soil analyses were completed to quantify the differences between the

two soil types. SOM was six times higher in the organic-rich soil than the sandy soil. Soil

moisture was about two times higher in the organic soil. Percent total carbon was 16

times higher in the organic soil compared to the sandy soil. Percent organic C was peak

adjusted but still found to be 0 for the sandy soil and was 1.60 for the organic soil. Lastly,

percent nitrogen was 0.29 times higher in the organic soil. Also, the groundwater used in

the experiment had an initial pH of 6.5.

The flow rates for the cores harvested on Day 3 were 66.95 ± 15.16 mL/day and

42.68 ± 13.49 mL/day for the sandy and organic core respectively (Fig. 5). This was

between 6 and 2.7 mL times higher than the target groundwater flow rate (25 mL per

day) with an estimated porosity of 0.5. For the sandy and organic cores harvested on Day

6, the flow rates were roughly identical but were also about 2.4 times faster than the

target groundwater flow rate (Fig. 6).

PPCP soil recoveries were used to quantify the amount of PPCPs lost in the

experiment for each soil type and the extraction blank was used to quantify the amount of

PPCPs lost in the extraction process. The amount of PPCPs recovered in the organic soil

recovery ranged from 15% for clofibric acid to 100% for estradiol. Sandy soil recoveries

ranged from 9.7% for clofibric acid and 100% for estradiol (Table 5). The extraction

blank showed between 8.2% recovery for clofibric acid and 100% recovery for estradiol

and triclosan. The greatest differences in the sandy and organic recoveries was found in 4

MB and triclosan with 17-20% more found in the sandy soil. However, for clofibric acid

and ibuprofen, a 6-10% greater recovery was found in the organic soil. Estradiol and

AHTN had the same recoveries for both sandy and organic soil. Extraction blank

recoveries were higher than soil recoveries for all PPCPs except clofibric acid. The 100%

recoveries for estradiol and triclosan in the extraction blank were adjusted because they

indicated over 100% recovery.

Large differences in extract color and amount of precipitate formed after dilution

with water was noticed between the two soil types during the extraction process. The

organic-rich soil extractant was vibrant green, while the sandy soil was golden yellow.

When 50 mL of water was added to the 0.5 mL solvent extracts before SPE loading, the

organic-rich soil extractant turned an opaque olive green color suggesting the presence of

carbohydrates and proteins precipitated and stuck on the inside of the flask.

The top of the cores harvested on the first day were analyzed to determine what

concentrations of PPCPs were present, if any, at the beginning of the experiment. In the top

sandy core harvested on the first day, we found triclosan, AHTN, and 4 MB present in the highest

concentrations, ranging from 690 to 1,525 ng/gdw (Table 6). Estradiol was present in smaller

quantities at 29 ng/gdw and clofibric acid and ibuprofen were not detected. In the top of the

organic core harvested on the first day, again, triclosan, AHTN and 4 MB were present in highest

amount between 176 and 563 ng/gdw, however the amounts were one order of magnitude lower

than the amounts found in the sandy core.

Then we compared the partitioning of PPCPs through out all sections of the sandy cores

and effluents harvested on the third and sixth day (Table 7). For the sandy core harvested on the

third day, 100% of clofibric acid and ibuprofen were found in the effluent collected on the first

day of the experiment. 84% of the hormone, estradiol, leached out in the effluent collected on the

first day similar to clofibric acid and ibuprofen. However, 7% of the compound was also found in

the top and middle of the core. 7% was also found in the effluent collected on day two and three.

90% of the fragrance additive: AHTN, 95% of the sunscreen additive: 4MB and 81% of the

antibiotic: triclosan, were found in the top and middle sections of the core.

In the sandy core collected on the sixth day, 100% of clofibric acid and ibuprofen were

found in the effluent in the first day (Table 8). This was identical to results found in the sandy

core harvested on day 3 reaffirming their migration into the effluent prior to the third day. On the

sixth day, AHTN showed evidence of migration between the third and sixth day with 70% in the

bottom layer of sediment compared to 5% found on the third day. No detectable levels of AHTN

were found in any of the effluents collected. Increased amounts of 4MB and triclosan were

recovered in the effluents collected on days 5 and 6, indicating their transition to the aqueous

phase.

When comparing the migration of PPCPs in the organic cores harvested on the third and

sixth day, we found a slower rate of leaching of clofibric acid and ibuprofen in core 6,

indicated by 66% and 85% found in the effluent collected on the first and second day and

34% and 15% detected in the effluent collected on the third and fourth day (Table 8).

Both AHTN and 4 MB showed migration from the third to sixth day, but no presence of

each PPCP was found in the effluents indicating no transition to the aqueous phase

throughout the duration of the experiment in the organic rich sediment. In contrast, both

triclosan and estradiol showed increased presence in the middle and bottom sections of

the core with 29% present for triclosan and 63% present for estradiol. The effluents also

indicated migration through the core and into the aqueous phase.

When compared the sandy and organic cores, we found similiar results for clofibric acid

and ibuprofen which indicated ~100% of these compounds leaching out the in the effluent

collected on the first day, with some noticeable retardation in the organic cores, indicated by 34%

and 15% of clofibric acid and ibuprofen found in the effluent collected on the third and fourth day

(Table 8). Differences in partitioning of AHTN, 4 MB, and triclosan were found in the organic-

rich core with 85-100% found in the top layer of the sediment compared to a 27-47% recovery in

the top layer of the sandy cores. Also, estradiol behaved differently in the organic sediment with

89% remaining in the sediment compared to only 8% recovered in the sandy soil.

Migration Rate Estimates

Migration rates were estimated using the data collected from cores harvested on

the sixth day. Rates were calculated by measuring the distance the compounds had moved

through the cores (inches) divided by the time this migration took (6 days). A minimum,

maximum and median rate was calculated for each compound by the shortest and longest

distances traveled and the point at which 50% of the compound is found both above and

below the core. Estradiol had the most variable migration rate throughout the core; it was

present throughout the cores and effluents (Fig. 7). The hydrophilic compounds, clofibric

acid and ibuprofen moved through the cores in one day so their migration rates are at

least 25 mL/day. The most hydrophobic compounds had the smallest ranges, and slowest

migration rates (0.16-3 mL/day). As Kow of each PPCP increased, the corresponding

migration rates decreased (Fig. 8)

Presence of PPCPs in Groundwater collected at Mashapaquit Creek

Detectable concentrations of estradiol, triclosan and 4 MB were found in the

groundwater sampled from the established well at concentrations of 3.5, 6.5 and 4.4

ng/mL respectively (Table 9). These numbers were one order of magnitude larger than

values found the previous year at established wells surrounding West Falmouth Harbor

similar to the well used to collect groundwater in this experiment. (Zhang et al., 2014).

Sediment in this area could have accumulated hydrophobic compounds. Testing un-

spiked soil for presence of compounds could further clarity. Additionally, these values pose

a potential explanation of recoveries greater than 100% for estradiol and triclosan. Also the

recovery rate was signicantly higher for 4 MB at 86% compared to 8%, 38% and 44% for

clofibric acid, ibuprofen and ATHN.

One essential component for analyzing the migration of PPCPs through soils that was not

addressed in this experiment is analysis of the collected sediments for presence of PPCPs before

adding compounds for migration analyses. Since we found significant concentrations of PPCPs in

the groundwater, this suggests that there is a strong possibility of measureable PPCPs present in

the sediments as well that may be contributing to higher recoveries found in this study.

E. Discussion

Effect of Hydrophobicity on Migration of PPCPs

Although this is a preliminary study, my data yielded consistent results in cores

harvested on Day 1, 3 and 6. As hydrophobicity of PPCPs increased, indicated by the Kow,

migration rates slowed through the soil and PPCPs were more likely to be retained in the

sediment rather than the effluent. This included compounds such as triclosan, AHTN and 4 MB.

More hydrophilic compounds (low Kow) such as clofibric acid and ibuprofen migrated

rapidly and were eluted from the cores in less than 24 hours. These compounds were

found almost exclusively in the effluents. This is explained by their high soluabiltiy in

water indicated by their low Kow. Estradiol, which was moderately hydrophilic, compared

to the other PPCPs used in the study, showed the majority of the compound present in the

effluent as well as detectable levels seen in the sediment. Thus, differences in migration

rate for compounds with varying hydrophobicity can be predicted from their varying

affinity for soil and water based on their chemical structure. This same result was found

by Gibson et al. (2010) when he explored the migration of PPCPs with varying

hydrophobicity through sewage sludge. Soil tends to be negatively charged which

facilitates interactions with positively charged molecules and Van der Waals interactions

(Gibson et al. 2010).

Effect of Soil Type on Migration of PPCPs

The effect of soil type on migration of PPCPs also yielded conclusive results. Migration

was retarded for all PPCPs in organic soil. This was more pronounced for hydrophobic

PPCPs which indicates that soil organic matter influences hydrophobic compounds more than

hydrophilic compounds, as seen by Chefetz et al. (2010) and Gibson et al. (2010). Porosity

differences of soil rich in organic matter and sandy soils may also play a role in the

hydrodynamic dispersion (Watson et al. 1998). Although a quantitative measurement of porosity

was not obtained for this experiment, we assumed that the porosity was greater for the sandy soil

based on grain size.

Overall Implications

Contamination of Grounwater Resources

Our study shows that the migration of PPCPs is highly dependent on the hydrophobicity

of each compound and the presence of soil organic matter. This means that there is a higher

potential for contamination downgrandient of wastewater inputs in areas with sandy soils, as

PPCPs will travel faster. As sandy soils make up the bulk of soils on Cape Cod, intial removal of

these compounds by advanced wastewater treatment is highly recommended to avoid

contamination of groundwater resources.

Concentrations of estradiol, triclosan and 4 MB in the FWTP plume were one order of

magnitude higher than values for estradiol and triclosan found by Zhang et al. (2014) in

established wells around West Falmouth Harbor (4 MB was not detected by Zhang et al. (2014)).

We cannot determine if Zhang et al. (2014) collected her samples from the same established well

as this study, but they are still signicantly higher than values found by Standley et al. (2008) in

the upper Cape Cod region (0.002 ng/mL and 0.16 ng/mL for β-estradiol and triclosan

respectively). The magnitude of these values suggests the accumulation of these compounds to

levels that may be harmful to fish and other organisms present in Mashapaquit Creek and West

Falmouth Harbor. Robertson et al. 2009 found that hep-1, a highly conserved gene resonsible for

regulating immunity in largemouth bass (Micropterus salmoides) and smallmouth bass

(Micropterus dolomieu) was compromised by the exposure of β-estradiol. The concentration of

triclosan found in the groundwater is one order of magnitude greater than 0.21 ng/mL, the net

effect concetration, which sets the limit at which no toxicity to river biofilm bacteria is observed

(Ricart et al. 2010).

Recommendations for Future Studies

This study addressed the mobility of PPCPs through varying soil types on Cape Cod.

However, to understand how these PPCPs interact with soils on a long-term scale, we must look

at their persistance in the environment. One way in which this could be studied could be to use an

experimental design used by Hchtoen et al. (1995) in which he contained sediment in

polyethelene boxes, spiked with PPCPs to analyze the persistance of various antibacterials over a

period of 18-300 days (Hallen-Sorenson et al. 1998). Peake et al. 2015 found that microbial

activity plays an important role in the degradation of these compounds. According to Quintana et

al. (2005), ibuprofen degraded cometabolically which suggests that it can be fully mineralized by

microbes.

Acknowledgements

I would like to specifically acknowledge my advisors Dr. Maureen Conte and JC Weber. Dr.

Conte helped me formulate a project and presentation that was understood by the general public.

JC Weber performed extraction analyses with me in the lab and help me analyze the data. I would

also like to give special thanks to Alice Carter who came up with creative ways for me to

calculate and graph migrations rates of PPCPs, among other things. Finally, I would like to thank

Kenneth Forman, Rich McHorney and Brecia Douglas for helping me obtain a groundwater flow

rate comparable to that on Cape Cod. I could not have done it without the guidance from these

individuals.

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Robertson, LC., Iwanowicz, LR., and Marranca, JM., 2009. Identification of centrarchid hepcidins and evidence that 17beta-estradiol disrupts constitutive expression of hepcidin-1 and inducible expression of hepcidin-2 in largemouth bass (Micropterus salmoides). 6: 898-907. Smith, J.A., Witkowski, P.J., and Fusillo, T.V., 1988. Manmade organic compounds in the surface waters of the United States--A review of current understanding. U.S. Geological Survey Circular 1007: 92. Standley, L.J., Ruthann, R.A., Swartz, C.H., 2008. Wastewater-Contaminated Groundwater as a Source of Endogenous Hormones and Pharmaceuticals to Surface Water Ecosystems. Environmental Toxicology and Chemistry 27:2457-2648. Ternes, A.T., Herrmann, N., Bonerz, M., Knacker, T., 2004. A rapid method to measure the solid-water distribution coefficient (Kd) for pharmaceuticals and musk fragrances in sewage sludge. Water Research 38: 4075-4084. Watson, K. K., and Jones, M. J., 1982. Hydrodynamic dispersion during adsorption in fine sand: 1. The constanst concentration case. Water Resources Research: An AGU Journal. 18: 91-100. Zhang, Y., Conte, M., and Weber, J.C., 2014. Occurrence and Reduction of Pharmaceuticals and Personal Care Products in Groundwater and Wastewater of Cape Cod, Massachusetts. SES Research Project, Marine Biological Laboratory, Wood’s Hole, Massachusetts, USA.

F. Tables Table 1: PPCP chemical structure, source and octanol water constants (Kow).

Table 2: Characterization of Soils Collected from Mashpaquit Creek. Sand Organic Soil Organic Matter 0.5% 3.0%

Soil Moisture 25.0% 46.7% Percent Total C 0.1% 2.5%

Percent Organic C BDL detection limit 1.6% Percent N 0% 0.3%

Table 3: PPCP Retention Times and Diagnostic Ions Used for Quantification on GC/MS

Compound Name Retention Time (min) Target Ion (m/z) Supplemental Target

Ion (m/z) Clofibric Acid 33.33 128 169

Ibuprofen 34.22 160 117 β-Estradiol 53.541 285 416 Triclosan 44.59 200 347

AHTN 39.57 243 159 4-Methyl-

Benzylidene 43.239 254 128 Retention time is the amount of time is takes for the PPCP to reach the GC/MS detector. The target ion is the ion in the mass spectra used to identify the PPCP. The supplemental was used to verify any inconsistencies.

Table 4: Slopes, Intercepts and R2 Values for PPCP Calibration Curves.

Clofibric

Acid Ibuprofen β-

estradiol Triclosan AHTN 4 MB Slope 29567 28612 39223 58597 51610 22327

Intercept -583616 -424388 -263991 -396511 -61813 -202032

R2 0.89 0.93 0.93 0.97 0.98 0.89

Table 5: Percentage of PPCP Spike Recovered in Soil Recoveries and Extraction Blank

    Clofibric  Acid  

Ibuprofen   Estradiol   Triclosan   AHTN   4  MB  

Organic  Soil     15%   39%   100%   52%   41%   43%  Sandy  Soil     9%   20%   100%   72%   41%   60%  Extraction  Blank    

8%   38%   100%   100%   44%   81%  

Table 6: PPCP Concentration (ng/gdw) found in the top section of the sandy core harvested on Day 1. Amount'in'Day'One'(ng/gdw)

Sand'Day'1 Clofibric'Acid Ibuprofen Estradiol Triclosan AHTN' 4MBTop 0 0 29 1526 823 690

Organic'Day'3 Clofibric'Acid Ibuprofen Estradiol Triclosan AHTN' 4MBTop 0 0 180 455 176 562

The gradient of blue to brown highlights on the name of the compound represents varying hydrophobicity; blue = more hydrophilic, light blue = moderately hydrophilic, and light brown = hydrophobic.

Table 7: Partitioning of PPCPs Through Sandy Cores Harvested on Day 3 and 6.

0%1$25%25$50'%50$75'%75$100'%

Key

Sand%Day%3 Clofibric%Acid Ibuprofen Estradiol Triclosan AHTN% 4MBTop 3% 47% 48% 27%

%Middle 5% 43% 47% 34%%Bottom% 8% 5% 30%

%Effluent%Day%1% 100% 100% 84%%Effluent%Day%2 6% 2% 4%%Effluent%Day%3 2% 2% 5%

Sand%Day%6 Clofibric%Acid Ibuprofen Estradiol Triclosan AHTN% 4MBTop 11% 12% 8% 21%

%Middle 10% 29% 22% 32%%Bottom% 47% 70% 34%

%Effluent%Day%1%and%2 100% 100% 54%%Effluent%Day%3%and%4 18% 2%%Effluent%Day%5%and%6 7% 10% 13%

The rows of the table that are highlighted in tan represent data collected from sections of the core. The rows in blue represent data collected from the effluents. The gradient of blue to brown highlights on the name of the compound represents varying hydrophobicity; blue = more hydrophilic, light blue = moderately hydrophilic, and light brown = hydrophobic. The gradient of red represents how high the percentage of PPCPs were found in a particular section or effluent (see key located above table).

Table 8: Partitioning of PPCPs Through Organic Cores and Effluent on Day 3 and 6.

0%1$25%25$50'%50$75'%75$100'%

Key

Organic(Day(3 Clofibric(Acid Ibuprofen Estradiol Triclosan AHTN( 4MBTop 23% 85% 100% 99%

(Middle 39% 7%(Bottom( 27% 6%

(Effluent(Day(1 100% 93% 1%(Effluent(Day(2 7% 4% 1%(Effluent(Day(3 7% 1% 1%

Organic(Day(6 Clofibric(Acid Ibuprofen Estradiol Triclosan AHTN( 4MBTop 23% 63% 92% 79%

(Middle 35% 16% 5% 21%(Bottom( 28% 13% 3%

(Effluent(Day(1(and(2 66% 85% 0% 2%(Effluent(Day(3(and(4 34% 15% 6% 2%(Effluent(Day(5(and(6 8% 2%

The rows of the table that are highlighted in tan represent data collected from sections of the core. The rows in blue represent data collected from the effluents. The gradient of blue to brown highlights on the name of the compound represents varying hydrophobicity; blue = more hydrophilic, light blue = moderately hydrophilic, and light brown = hydrophobic. The gradient of red represents how high the percentage of PPCPs were found in a particular section or effluent (see key located above table).

Table 9: Detectable Concentrations of PPCPs found at Study Site on Mashapaquit Creek.

Compound Concentration (ng/ml)

Estradiol 3.56

Triclosan 6.52

4 Methylbenzylidene 4.35

G. Figures

Figure 1: Map of Sampling Site. The green arrow represents the direction of flow of the West Falmouth Treatment Wastewater plume. The red arrow represents the study site.

Figure 2: Experimental Design. Consisted of 12 cores (6 with each soil type), 35 cm in length and 1.25 cm in diameter. The groundwater was stored in a Mariotte bottle which supplied the needed pressure to pump groundwater at 25 cm/day. Groundwater was pumped through 0.38 mm polyethylene tubing and into a 27 gage needle supported by a red septum. Effluent bottles were collected every 24 hours to calculate variation in flow rates through out the experiment.

Figure 3: Overview of Analytical Method Used to Extract PPCPs.

Figure 4: Standard Calibration Curves For Each PPCP. The numbers to the right of the title are the target ions used for quantification.

Figure 5: Flow Rates for Cores harvested on Day 3.

Figure 6: Flow Rates for Cores Harvested on Day 6.

Figure 7: Range of Migrations Rates in Sandy and Organic Cores Harvested on Day 6. Error bars show maximum and minimum rates and blue bar shows the middle 50% of the compound.

Figure 8: A Closer look at the migration of the hydrophobic compounds. Error bars show maximum and minimum rates and blue bar shows the migration rate range for 50% of each compound.

Figure 9: Comparison of Log Kow and Migration Rates.

Figure 8: A Closer look at the migration of the hydrophobic compounds. Error bars show maximum and minimum rates and blue bar shows the migration rate range for 50% of each compound.

Figure 9: Comparison of Log Kow and Migration Rates.