Use of detergent additive, linear alkylbenzene sulfonate, as an indicator of wastewater input
to Oyster Pond, MA
Sarah Erskine
Wheaton College, Norton, MA
Maureen Conte
Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA
J.C. Weber
Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA
Semester in Environmental Science 2013
Abstract
Linear alkylbenzene sulfonate (LAS) is a common surfactant used in detergents
worldwide. A sediment core from Oyster Pond in Falmouth, Massachusetts was analyzed for
presence of LAS to determine if it could be used as an indicator of wastewater input to Oyster
Pond. Using the GC/MS, concentrations were found and compounds were identified. Septic
system samples were taken from a Standard Title V system to determine how much LAS would
be introduced to groundwater. No LAS was found in the most recent sediments due to septic
system treatment, but older sediment has concentrations high enough to be used as an
indicator.
Key Phrases
Wastewater Input; Anionic Surfactant; Oyster Pond; Linear Alkylbenzene Sulfonate; Septic
System
Key Words
Surfactant; LAS; Sediment; Indicator; Wastewater
Introduction
Surfactants are largely used worldwide in detergents and cleaning products. They have
hydrophilic heads and hydrophobic tails, their alignment in mixtures of two liquids decreases
surface tension and their structure helps bind to “dirty” compounds such as greases. Linear
alkylbenzene sulfonate (LAS) is the main surfactant used in detergents. LAS is an anionic
surfactant, with a sulfonate (R-SO3-) head and a hydrophobic tail with 10-14 carbon atoms (Fig.
1) (León, González-Mazo, Pajares, & Gómez-Parra, 2001). It biodegrades by ω-oxidation at a
terminal methyl group and β-cleavage (Eganhouse, Blumfield, & Kaplan, 1983).
LAS’s common use in households guarantees its presence in domestic wastewater.
Some LAS is removed during wastewater treatment, but even treated water contains significant
concentrations; an even higher concentration is present after treatment in septic systems
(McAvoy, White, Moore, & Rapaport, 1994; Tabor & Barber, 1996). LAS is further removed as it
travels through the aquifer and groundwater (Thurman, Barber, & LeBlanc, 1986). Since LAS
stays in the environment at measurable concentrations for decades, it can be helpful to look at
its presence in aquatic systems with significant wastewater input; in addition, there is a
possibility it could be used as an indicator of this wastewater input (Reiser, Toljander, Albrecht,
& Giger, 1997).
Concentration and Identification of LAS is possible through gas chromatography mass
spectroscopy (GC/MS), and provides reliable data but analysis by high performance liquid
chromatography (HPLC) is preferred because there is less possibility of error introduced during
derivatization and clean up (Ding & Fann, 2000; Kikuchi, Tokai, & Yoshida, 1986).
Methods
Site Description
Oyster Pond is a shallow pond located in Falmouth, MA (Fig. 2 ). It is composed of three
kettle ponds and has a total surface area of approximately 25 hectares (Wright-Pierce, 2013). It
is surrounded by residential homes and has a condominium complex on the northern end with
62 units and 27 septic systems. All buildings in the Oyster Pond watershed use septic systems,
none are on a sewer system (Wright-Pierce, 2013). A sediment core of 32cm was taken from
the northern end of the pond (Fig. 2). The sediment core was dated using the sedimentation
rate of 0.88cm/year in a nearby pond in Falmouth with similar physical characteristics (Keafer,
Buesseler, & Anderson, 1992).
Three, four liter, septic system samples were collected from a Standard Title V septic
system at the Massachusetts Alternative Septic System Test Center (MASSTC) from the main
influent channel, the septic tank, and the effluent from the leaching field. The system is dosed
at 450 gallons a day and the tank is 1000 gallons, giving a retention time of about 2.22 days.
The system was being treated with a stress test of three was loads created as a large
cycle of soap (Arm and Hammer Power Laundry Detergent, Clean Burst) and non-chlorine bleach
(Clorox 2 Stain Fighter and Color Booster, Original Scent) (Fig. 3). The laundry loads are created
as a large cycle with 12 gallons wash and 24 gallons rinse with about 108 gallons total for the
three loads. 108.5 grams of soap and 99 grams of non-chlorine bleach were added per large
load. These loads are added to a distribution system tank between the main influent and the
septic tank; this distribution system tank could not be sampled. The treatment was started
three days prior to sampling with a total of two large cycles, one on the morning of the day the
stress test was started, and one about 50 hours prior to sampling.
Blanks and Standards for Standard Curve
I created two blank samples to run with my septic and sediment samples: one blank
sample of internal standard and one standard LAS mix. I spiked the LAS mix with 10µL of C8-LAS
internal standard. These two blanks were passed through the SAX column, derivatized, and
passed through all the columns for cleanup and the final filter in clean up. I ran these blanks on
the GC/MS with the rest of my samples.
I created five standards to use to make a standard curve. I filled 40mL glass vials with
the following volumes of LAS mix: 0.5mL, 1mL, 2mL, 3mL, and 4mL. The LAS was put in a drying
oven to evaporate off water in the mixture. I added 10µL of internal standard and transferred
to clean 13mm Pyrex test tubes with methanol. The standards were evaporated to dryness in
Savant on high heat for 20 minutes, reacted with 200µL thionyl chloride, and kept in a 95oC oven
for one hour. After the standards cooled to room temperature I added 200µL trifluoroethanol
and 200µL pyridine, and then put them in the 95oC oven to react for 20 minutes. I evaporated
the standards in the Savant for 20 minutes at medium heat to reduce volume to about 50µL. I
passed the standards through the alumina column with hexane/ethylacetate (1:1) and
evaporated to dryness. I resuspended the standards in 100µL methylene chloride and
transferred to a 2mL vial where I blew down to a volume of 50µL using zero nitrogen gas. I
transferred the standards to screw top v-vials and ran them on the GC/MS using the same
temperature program as with the samples.
Preparation for Extraction
I sectioned the Oyster Pond sediment core in one centimeter sections (Fig. 4). The top
several centimeters of the core were lose and had to be siphoned off. The sediment became
progressively firmer and denser as depth increased. Only 23 sections were obtained from the
32 centimeter core, meaning the sections were not exactly 1 centimeter, equation 1 was used to
adjust depths. I selected eight sections (1.4cm, 2.8cm, 4.2cm, 5.6cm, 13.9cm, 15.3cm, 23.7cm,
and 32.0cm) to represent the most recent sediments, the oldest sediment, and some
intermediates where the core looked somewhat different in color or texture. I loosely packed
these sections into labeled 40mL glass vials allowing for enough space for the expansion of
water when it froze. I placed small square of combusted aluminum foil over the top of the vial,
and punctured holes with a 23 gauge needle and screwed on the caps. I placed the samples in a
-30oC freezer.
After approximately 21 hours, I removed the samples from the freezer, and placed the
samples in the freeze drier. If any vials were cracked, I put them in an opened clean plastic bag
and then into the freeze drier vacuum flasks. To reduce any mixing of sediments from samples,
all vials from the same depth were put into the same flask with another set of samples from the
next depth (the 1.4cm vials with the 2.8 cm vials). I freeze dried the samples 48 hours.
After removing dried samples from the freeze drier, I combined vials of the same depth
on combusted aluminum foil and homogenized the sample. I removed roots, leaves, rocks, and
still frozen sediment from the sample, homogenized, and returned to the original 40 mL vials,
combining into as few vials as possible. I took care to not fill the vials more than half way and
took all samples’ weights. I kept a small subsample for CN and N15 analysis.
I filtered 50-500mL of the septic system samples through 47mm GF/F filters under
vacuum, filtering the cleanest sample first. I stored filtrate and filters in separate labeled 40mL
glass vials. I added 20mL methanol to the filters.
Extraction
I added 30mL of methanol to each vial with weighed out, freeze dried, sediment and
added a total of 10µL of C8-LAS internal standard to each depth. I ultrasonicated for 30 minutes
at an output power of 22%, centrifuged for 10 minutes at 1500rpm, and pipetted off methanol
taking care to not disturb the sediment and put the methanol into clean, labeled 40mL glass
vials. I added 20mL of methanol to the sediment and repeated untrasonication for 10 minutes
at an output of 22% and centrifuged for 10 minutes at 1500rpm, and finally pipetted off the
methanol and combined with the first 30mL of methanol.
Using a strong anionic exchange cartridge from Agilent Technologies, I rinsed the column
with 5mL of methanol, making sure once the column did not dry at any point once it was wet
and to not exceed a drip rate of 2mL per minute (about three drops per second). I passed the
sample through and collected in a new labeled 40mL glass vial, rinsed with 2mL methanol and
collected into the same vial. To collect the LAS that was kept in the column, I passed through
5mL of 5% HCl in methanol and collected in a 13mm glass centrifuge tube.
I added 100µL of C8-LAS internal standard to the septic system filters in methanol. The
filters were untrasonicated at 22% output power for 30 minutes. After I pipetted off the
methanol into a clean labeled 40mL glass vial, I add 10mL of methanol to the filters and
repeated untrasonication for 30 minutes at 22% output power and the methanol pipetted off
and combined with the first 20mL of methanol.
The filtrate was extracted on C18-Disks from Supelco Inc. following their instructions
with slight modification (Supelco, 1994). I cleaned the disks by passing through 5mL of
methanol and drying for five minutes under vacuum. I then conditioned the disks under low
vacuum with 5mL methanol and 5mL Milli-Q water. A subsample was not passed through the
disk and kept for nitrate analysis. I dried the disk under vacuum for five minutes and extracted
analyte with 15-20mL of methanol, collected in a pear flask, and transferred to a clean labeled
40mL glass vial.
Derivatization
To derivatize my samples I followed methods by Reiser, Toljander, and Geiger (1997) and
Trehy, Gledhill, and Robert (1990) with slight modifications. I added 200µL of thionyl chloride to
samples and flushed with zero nitrogen gas for 30 seconds, and capped under nitrogen.
Samples were kept in 95oC oven for one hour and cooled to room temperature. I added 200µL
trifluoroethanol and 200µL pyridine, flushed with zero nitrogen gas for 30 seconds, and capped
under nitrogen. I kept samples in a 95oC oven for 20 minutes then evaporated down to about
50µL in Savant at medium heat for about 20 minutes.
Cleanup
Following Reiser et al. (1997) and Trehy et al. (1990) I cleaned up my samples. Using a
13cm column with approximately 1.5g alumina, I passed through my sample with 2mL
hexane/ethylacetate (1:1) and collected in a clean and labeled 13mm glass centrifuge tube. I
evaporated the samples to dryness in Savant under medium heat for 10-15 minutes then
resuspended in 50µL hexane/ethylacetate (1:1). Samples were then passed through a 13cm
column of 200µg copper powder on a bed of 0.5g alumina. I passed through my sample with
2mL hexane/ethylacetate (1:1) and collected in a clean and labeled 13mm glass centrifuge tube.
I evaporated to dryness in Savant under medium heat for 10 minutes and resuspended in 50µL
hexane. Samples were then passed through a 13cm column of about 1.0g silica gel with 5mL
hexane/ethylacetate (9:1). Samples were evaporated to dryness in Savant at medium heat for
30 minutes. I resuspended samples in 500µL methylene chloride and passed through fritted
extraction tubes with 4mm Millex-FH filters and collected into a labeled 2mL vial. I blew down
the samples with zero nitrogen gas and resuspended in 50µL methylene chloride and
transferred to 100mL screw top V-vials for analysis on GC/MS.
GC/MS
I injected 1-3µg of sample onto the GC/MS and ran a temperature program of 50oC for
two minutes, ramped to 200oC at 10oC per minute, then ramped to 320oC at 6oC per minute and
held at 320oC for 15 minutes.
Identification and Quantification
I used FID to make a standard curve from my standard samples and took
the average of the linear regression for each individual compound I could identify (Equation 2).
The standard deviation between the slopes and intercepts was small enough that it did not
seem to make a difference if I used, for example, the 2-C10 LAS to find the concentration of a
1/3-C11 LAS, this also allowed me to have a way to find concentrations of compounds that I did
not have in my LAS mix but did have in my sediment or septic samples.
I used the areas of the ion 91 peaks as my areas for LAS peaks with the
same retention time, since 91 is an ion in an LAS compound. I used the internal standard to
normalize the areas of the 91 peaks then used the standard curve equation to convert the area
to concentration.
To be able to compare concentrations from sediments to concentrations from a septic system I
used CHN analysis to put concentrations in terms of grams of Carbon.
Results
LAS in Septic Systems
The septic system shows an increase in nitrate, with a change in concentration of three
orders of magnitude from influent to effluent (Table 1). LAS does not show an increase or
decrease in total concentration from influent through to effluent. Total LAS is highest in influent
and lowest in the tank (Fig. 5). Partitioning between dissolved LAS and LAS bound to suspended
particulates are similar between influent and effluent, and a much higher percentage of
suspended particulate attributed to the partitioning in the tank (Fig. 5). Chain length
distribution for dissolved LAS shows similar patterns between the influent and effluent with C13
as the least abundant chain length and C11 and C12 as the highest (Fig. 6). The tank shows a
different distribution compared to the influent and effluent and favors shorter chain lengths
with a highest abundance of C10 then C11 and C12 and no C13 (Fig. 6). All septic system
samples show the same general pattern of distribution across chain lengths for suspended
particulate-bound LAS with relatively even percentages of C10, C11, and C13 and the majority of
LAS from C12 (Fig. 6).
LAS in Sediments
Total LAS in sediments shows no LAS until sample depth 13.9cm (approximate year 2001)
with a concentration ranging from 2.74 to 17.06µgLAS/gC between 2001 and 1985 (Fig. 7). A
chain length of 12 carbons is the highest concentration across most depths with a 13 carbon
chain as the lowest concentrations (Fig.8). Distribution of chain length is similar between the
years 1992 and 2000 and similar between the years 2001 and 1985 (Fig. 9). 2001 and 1985 have
a higher percentage of shorter chain lengths compared to 2000 and 1992, where 2000 and 1992
have the majority of their chain lengths C12 (Fig. 9). The distribution of chain length in 2001 and
1985 is closer to the distribution of chain lengths seen in the septic tank sample and the
distribution in 2000 and 1992 have a distribution closer to that of the septic influent and
effluent (Fig. 6, 9). There is no obvious pattern between distributions of group position in the
sediment samples (Fig. 10).
Stable Isotopes
N15 and C13 isotope analyses run on the septic system samples show lightest carbon
and heaviest nitrogen in the influent and heaviest carbon and lightest nitrogen in the effluent
with the tank’s values in between (Fig. 11). I am hesitant about the negative δ15N values (Fig.
11) . The sediments shows isotopic values more similar to fresh water in the more recent
sediment and more similar to saltwater in the older sediments (Fig. 12). There is no similarity
between the septic tank stable isotopes and the sediment stable isotopes (Fig. 11, 12). The
nitrogen shows a pattern somewhat like the pattern seen in total LAS concentration through
depth (Fig. 7, 13). There is no obvious correlation between the 15N values for the septic system
and Oyster Pond (Fig. 11, 13).
Discussion
LAS in septic systems
My results show the important partitioning between dissolved LAS and LAS bound to
suspended particulates at different stages in a Standard Title V system (Fig. 5). I expect this
partitioning between the samples to be due to the characteristics of the stages of the septic
system. Since only aqueous samples were taken, any LAS in the sludge in the septic tank was
not accounted for, the only particulate-bound LAS was suspended particulate. Doing this
experiment again it would be insightful to look at the LAS content in the sludge or biofilm. I
believe the characteristics of the way water flows through the septic system is also an important
factor in the partitioning seen between the samples. Influent and effluent have more water
movement so there is a higher chance of LAS staying dissolved in the water rather than
adsorbing onto particulates where as in the tank the water becomes more stagnant and solids
separate out of the wastewater. Stagnant water and this separation between solids and liquids
are most likely the reasons why we see a higher percentage of suspended particulate bound LAS
in the tank. I believe the higher presence of biofilm in the septic tank is the reason for the lower
concentration of LAS because LAS biodegrades much more quickly in the presence of biofilm
(Takada, Mutoh, Tomita, Miyadzu, & Ogura, 1994). Once again I think it would be helpful to see
the LAS presence in the sludge or the biofilm to support this theory and to see if it could make
up for the drop in total LAS concentration seen from the influent to the tank and the increase
from the tank to the effluent (Fig.5).
I think water flow characteristics could also be the reason for the difference in chain
length distribution between the septic samples (Fig. 6). According to the “distance principle,”
LAS compounds with longer chain lengths with break down more readily than those with shorter
chain lengths (Swisher, 1987). With the higher presence of biofilm and solids in the septic tank,
I assume that more LAS is degrading, meaning a lower presence of longer chains.
LAS in sediments
Further investigation into the absence of LAS in the upper sediments led to the discovery
that Treetops Condominiums treats their 27 septic systems monthly with the product Septic
Doctor Tablet from Caldwell Environmental (Fig. 7). This product guarantees the digestion of
greases and other problem wastes, and since LAS is strongly associated with greases and has a
similar molecular structure, I believe that the tablets also degrade LAS (Fig. 14) (Caldwell
Environmental Inc., n.d.). Another core should be sampled with sections between 5.6 and
13.9cm to see tablet addition’s direct effect.
Chain length concentrations through depth increases and decreases in the same pattern
indicating the differences between years are not a product of analysis (Fig. 8). I believe that
water flow could once again be the reason for the observed distributions (Fig. 9). Increased flow
or flushing of Oyster Pond could cause distributions similar to septic tank influent and effluent
where longer chains are more abundant than shorter chains (Fig. 6). Large storms occurred in
1991 and 1999 and could be the reason for the distributions seen in 1992 and 2000.
Distribution of location of benzene group did not show any discernable patterns (Fig.10).
Stable Isotopes
The close similarity between the most recent sediment and its difference compared to
the deeper sediments shows that the Septic Doctor Tablet could be altering 13C input to Oyster
Pond, but it does not seem like it has significantly altered the 15N input (Fig. 11, 12). The similar
pattern seen between the total LAS concentration and the 15N values shows a possible
correlation between LAS and wastewater input since 15N is often used as an indicator (Fig. 7, 13)
(Valiela, 1995).
Conclusions
With Treetop Condominiums’ septic system treatment, LAS cannot be used as an
indicator of wastewater input to Oyster Pond for recent sediment, but high enough
concentrations of LAS are present in deeper sediments and can be used as an indicator. Further
research should be done with a deeper core and historical data of water use to determine an
equation of converting the concentration of LAS in sediments to what wastewater input would
have been.
Acknowledgements
I thank my advisors Maureen Conte and J. C. Weber for their guidance and help
throughout the project. I thank Richard McHorney, Becky Leone, Lauren Wind, Kelsey Gosselin,
and Tyler Ueltschi for help collecting and sectioning my sediment core. I thank Ken Foreman for
running Semester in Environmental Science (SES). I thank Wendi Buesseler and the rest of the
Oyster Pond Environmental Trust (OPET) for help finding a sampling location and putting the
pieces together. I thank Keith Mroczka and the Massachusetts Alternative Septic System Test
Center (MASSTC) for access to and endless information on septic systems. I thank Fiona Jevon,
Alice Carter, and Sarah Nalven for countless advice. I thank Wheaton College. I thank the
Marine Biological Laboratory and the Ecosystems Center for funding and use of equipment.
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Figures and Tables
Figure 1. Linear alklybenzene sulfonate (LAS) is an anionic surfactant with a hydrophilic
head and hydrophobic tail of 10-14 carbon atoms where x+y=n and n+2 is the
length of the carbon tail.
Figure 2. Oyster Pond, Falmouth, MA. Sampling site marked by a yellow circle and the
approximate location of Treetops Condominiums marked by a red outline. Map
courtesy of Google Maps.
Figure 3. Soap and non-chlorine bleach used for stress test of septic system sampled from
MASSTC.
Figure 4. Core sectioning was done by pushing the sediment core up into an empty core
(A) and using a spackling knife to cut and remove one centimeter sections by
sliding the spackling knife between the sediment core and the empty core (B).
The sectioned sediment was then moved from the spackling knife to a labeled
40mL glass vial (C). If sediment was not solid enough, the centimeter sections
were siphoned off.
Figure 5. Partitioning between total concentrations of LAS compounds in aqueous samples
from a septic system as dissolved LAS and LAS bound to suspended particulates.
Figure 6. Distribution of LAS compounds based on carbon chain length. Dissolved LAS in
aqueous samples from septic system (A) and suspended particulate bound LAS in
aqueous samples from septic system (B).
Figure 7. Concentrations of total LAS compounds versus depth with approximate age of
sediment based on a sedimentation rate of 0.88cm per year.
Figure 8. Concentration of LAS compounds based on carbon chain length in sediments of
approximate years from Oyster Pond core.
Figure 9. Distribution of LAS compounds based on carbon chain length in sediment core
from Oyster Pond.
Figure 10. Distribution of LAS compounds in years based on carbon chain length and
position of benzene sulfonate group identified in mass spectrum from GC/MS.
Figure 11. Stable isotope composition of aqueous septic system samples.
Figure 12. Sediment stable isotope values from Oyster Pond sediment core. Data points are
labeled with depth and approximate age of sediment.
Figure 13. Stable isotope 15N through depth of Oyster Pond sediment core.
Figure 14. Grease and LAS molecules join together because of their similar structures.
Table 1. Nitrate concentrations of aqueous samples from a Standard Title V septic system.
Equation 1. actual depth = section*(32/23)
Equation 2. y = 0.00002088x + 0.00123941
Figure 1. Linear alklybenzene sulfonate (LAS) is an anionic surfactant with a hydrophilic head
and hydrophobic tail of 10-14 carbon atoms where x+y=n and n+2 is the length of the carbon
tail.
Figure 2. Oyster Pond, Falmouth, MA. Sampling site marked by a yellow circle and the
approximate location of Treetops Condominiums marked by a red outline. Map courtesy of
Google Maps.
Figure 3. Soap and non-chlorine bleach used for stress test of septic system sampled from
MASSTC.
Figure 4. Core sectioning was done by pushing the sediment core up into an empty core (A) and
using a spackling knife to cut and remove one centimeter sections by sliding the spackling knife
between the sediment core and the empty core (B). The sectioned sediment was then moved
from the spackling knife to a labeled 40mL glass vial (C). If sediment was not solid enough, the
centimeter sections were siphoned off.
Figure 5. Partitioning between total concentrations of LAS compounds in aqueous samples from
a septic system as dissolved LAS and LAS bound to suspended particulates.
Figure 6. Distribution of LAS compounds based on carbon chain length. Dissolved LAS in
aqueous samples from septic system (A) and suspended particulate bound LAS in aqueous
samples from septic system (B).
A B
Figure 7. Concentrations of total LAS compounds in Oyster Pond core versus depth with
approximate age of sediment based on a sedimentation rate of 0.88cm per year.
Figure 8. Concentration of LAS compounds based on carbon chain length in sediments of
approximate years from Oyster Pond core.
Figure 9. Distribution of LAS compounds based on carbon chain length in sediment core from
Oyster Pond.
Figure 10. Distribution of LAS compounds in years based on carbon chain length and position of
benzene sulfonate group identified in mass spectrum from GC/MS.
Figure 11. Stable isotope composition of aqueous septic system samples.
Figure 12. Sediment stable isotope values from Oyster Pond sediment core. Data points are
labeled with depth and approximate age of sediment.
Figure 13. Stable isotope 15N through depth of Oyster Pond sediment core.
Figure 14. Grease and LAS molecules join together because of their similar structures.
Influent Tank Effluent
NO3 (µM)
0.0612 0.348 8.98
Table 1. Nitrate concentrations of aqueous samples from a Standard Title V septic system.
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