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Concentrations of Synthetic Fragrance Chemicals in Aquatic Systems Chelsea Burns and Kayla Tripp SURE Research: Summer 2011

Research Mentor: Sarah Rubinfeld

ABSTRACT Synthetic fragrance chemicals are present in aquatic systems due to discharge from wastewater treatment plants. Four specific chemicals, musk ketone (MK), musk xylene (MX), galaxolide (HHCB), and tonalide (AHTN), have been found in water, sediment, and aquatic organisms in several studies worldwide. These chemicals have the potential to cause negative health problems, leading scientists to discover new ways to rid them from aquatic environments. Water and sediment samples were acquired and the chemicals were extracted, condensed, and cleaned based on EPA methods. A GC/MS was used to analyze the samples and a calibration curve was derived to determine unknown concentrations in spiked samples. The R2 values for the MK and MX trendlines were 0.9718 and 0.9635, respectively. The R2 values for the HHCB and AHTN trendlines were 0.998 and 0.9922, respectively. A method detection limit (MDL) was calculated for each chemical to verify the lowest measureable concentration. The MDL for MK, HHCB, and AHTN was 0.106, 0.019, and 0.022 ug/L, respectively. The nitromusk fragrance chemicals, MK and MX, were found to have a percent recovery below 100%; the polycyclic fragrance chemicals, HHCB and AHTN, were found to have a percent recovery above 100%. INTRODUCTION

Aquatic systems have a variety of chemicals present; some are naturally-occurring and

others are created artificially. Water with products containing synthetic chemicals is sent to wastewater treatment plants through household drainage systems. Some of these man-made chemicals enter the environment through the effluent, or discharge point, of wastewater treatment plants. Wastewater treatment plants (WWTPs) in the United States are not meant to rid wastewater of chemicals, but they end up eliminating approximately 95% of them (Simonich et al., 2002). The remaining 5% of the chemicals get released into larger bodies of water, such as lakes and rivers.

The synthetic musk chemicals shown in Figure 1 provide the fragrance for many shampoos, cosmetics, and other personal care products. Nitromusk (NM) fragrance chemicals such as musk ketone (4-tert-butyl-2,6-dimethyl-3,5-dinitroacetophenone) and musk xylene (5-tert-butyl-2,3,4-trinito-meta-xylene) and polycyclic musk (PCM) fragrance chemicals such as HHCB (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-y-2-benzopyran) and AHTN (7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronapthalene) have become the focus of increased research over the past several decades as the worldwide population increases. The general structure of both nitromusk and polycyclic musk compounds includes benzene rings with attached carbon groups. These carbon benzene rings make the compounds hydrophobic. HHCB has been named a high-production-volume chemical, meaning more than 1 million pounds of the chemical is produced or imported each year in the United States (Peck et al., 2006).

     

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Musk Xylene Musk Ketone Tonalide Galaxolide

Figure 1: Chemical Structures of Synthetic Musk Compounds

Because of their hydrophobic properties, nitromusk and polycyclic musk fragrances can bind to sediment in aquatic systems. The accumulation of musk fragrances in sediment poses a threat to organisms that ingest it. HHCB and AHTN are listed on the Toxic Substances Control Act (TSCA) Inventory (Peck and Hornbuckle, 2004), meaning the chemical has the ability to harm the environment. The material safety data sheets (MSDS) state that both nitromusk and polycyclic musk fragrance chemicals can cause harm to aquatic life if ingested (Sigma-Aldrich). Polycyclic musks are more toxic to cells than nitromusk fragrance chemicals (Schnell et al., 2009). On the other hand, nitromusk fragrance chemicals are linked to other negative phenomena such as drug resistance in aquatic organisms (Matamoros and Bayona, 2006) as well as the induction of toxic liver enzymes (Schnell et al., 2009). Nitromusk and polycyclic musk fragrances are organic wastewater contaminants, which may potentially cause cancer, adverse physiological effects, antibiotic resistance, and reproductive impairment in humans and other animals (Kinney et al., 2006).

Other regions, such as Japan and Europe, have dedicated more research to the fate of musk fragrance chemicals. The concentrations of these chemicals have been measured in water since their initial detection in the Tama River near Tokyo, Japan in 1981 (Buerge et al., 2003). Concentrations of HHCB and AHTN were 0.0006-0.0042 mg/L in sewage effluent from various bodies of water in Europe (Ternes, 1998). Researchers in Switzerland have found average concentrations of 5 ug/kg for musk ketone and 30 ug/kg for musk xylene in dry soil (Berset et al., 2000). In the United States, Lake Michigan water was found to have an average concentration of 0.0000047 mg/L and 0.000001 mg/L for HHCB and AHTN, respectively (Peck and Hornbuckle, 2004).

The primary objective of this study was to develop a series of methods to measure the concentrations of fragrance chemicals in aquatic environments. Using Dr. Rubinfeld’s previous methods as a basis for the current experiment, processes such as extraction, clean-up, and analysis using a GC/MS were performed. Dilutions were created from known nitromusk and polycyclic musk standards and then used to produce calibration curves. The calibration curves were useful in determining the method detection limit (MDL) and unknown concentrations in field samples. Method detection limit experiments were performed to determine the lowest measurable concentrations. Percent recoveries were determined for both musk fragrance chemicals to test the reliability of the methods being performed. This was achieved by performing spike recovery experiments where known concentrations of the standard musk chemicals were added to samples of deionized water and sand. The field samples taken from the Pike River on Carthage College campus were hypothesized to contain little to no musk fragrance

     

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chemicals because the sample site is not located near the discharge point of a wastewater treatment plant. METHODS Sample Collection Two water and two sediment samples were collected in one liter amber glass bottles and 16 oz amber glass jars, respectively. The containers were dark-colored to prevent any chemical reactions with sunlight. These samples were obtained from the Pike River on the campus of Carthage College. The first sample site was approximately 0.1 miles from the west entrance of the David A. Straz Center at Carthage College, as seen in Figure 2 for exact location.

Figure 2: Map of Sample Collection Site. The big yellow “X” symbolizes the David A. Straz Center and the small red “X” symbolizes the location of the water and sediment sample collection.

The water samples were collected approximately two feet from shore by submerging the one liter glass bottle. When collecting the soil samples from the river, a small trowel was used to scoop up the sediment from underneath the water sampling location. The sediment sample was placed directly into the glass jar. Contamination was avoided by wearing non-powdered, nitrile gloves during sample collection. The water and sediment samples were placed directly into their corresponding containers to reduce transfer contamination. These samples were then transported to the Carthage College biochemistry lab. The sediment sample was allowed to dry for seven days on an aluminum foil tray in a fume hood. The entire contents of the glass jar were transferred into the tray using metal spatulas. The aqueous sample was placed in a freezer for one week until used for extraction.

     

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Sample Extraction The musk fragrance chemicals were extracted from the water sample following procedures based on EPA method 3510C. Extraction is necessary in order to target the specific fragrance chemicals being studied. Water samples were collected from the field as specified in the previous section. Aqueous samples were also created in the lab and used to perform spike recovery and method detection limit experiments. These aqueous samples were created using one liter of deionized water spiked with a known concentration of musk chemical standard. For both the field and lab aqueous samples, 40 mL of dichloromethane (DCM) was measured using a 50 mL graduated cylinder and was added to a separatory funnel containing one liter of aqueous sample. The separatory funnel was shaken for two minutes in ensure mixture of the aqueous and organic (DCM) layers allowing the chemicals to move from the water to the DCM. The organic and aqueous phases were allowed to separate for a minimum of 15 minutes. The organic DCM layer was drained into a 125 mL Erlenmeyer flask. Anhydrous sodium sulfate was added to the flask to remove any excess water. The musk fragrance chemicals were extracted from the sediment sample following procedures based on EPA method 3550B. Sediment samples were collected from the field site as described in the previous section. Spike recovery and method detection limit experiments were performed using sediment that was not obtained from a field site. Instead of using sediment from the field site, ~5g of clean sand was purchased and was spiked with a known concentration of musk chemical standard. Anhydrous sodium sulfate was added to the sediment samples to remove any excess water. For both the field and lab sediment samples, a Qsonica Ultrasonic Processor was used for 6 minutes in pulses of 15 seconds on vibration and 15 seconds off vibration to disrupt the sediment particles. A mixture of 1:1 hexane: acetone was used to separate the chemicals from the sediment during the sonication process. Since acetone mixes well with water, it was used to pick up any excess water, including chemicals, out of the sediment pores that the sodium sulfate may have missed. Hexane does not mix well with water and cannot enter the pores as easily; therefore, a combination of both chemicals was necessary. Following sonication process, the sample was allowed to filter into an Erlenmeyer flask. Anhydrous sodium sulfate was added to the sample during the filtration process to remove any final aqueous product. The extraction process for both aqueous and sediment samples was repeated a total of three times and the extracts were combined into a single flask. The liquid extract was concentrated to ~20 mL using an Organomation N-EVAP 111 nitrogen evaporator and was transferred into a 40 mL vial. The DCM solvent was then exchanged to hexane and concentrated to ~1 mL. Sample Clean-up The extracts from the aqueous and solid samples were passed through a florisil extraction cartridge to remove unwanted chemicals that might interfere with analysis. Each cartridge was rinsed with 10 mL of hexane in an Alltech 12-port vacuum manifold before adding the extract. The first elution solvent was 5 mL of hexane and was used to rid the sample of any chemicals other than the fragrance chemicals. The interfering chemicals passed through the florisil cartridge with hexane and were disposed of in a waste beaker. The second elution solvent was 5 mL of DCM which rinsed the fragrance chemicals from the florisil cartridge into a conical vial. Using a nitrogen evaporator, the DCM fraction in the conical vial was concentrated to ~200 uL.

     

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During the concentration process, the DCM solvent was exchanged to hexane using three 1 mL portions of hexane. Sample Preparation A 250 uL NS Target chromatography syringe was used to transfer the ~200 uL sample from the conical vial to a glass autosampler vial containing a 250 uL vial insert. The conical vial was rinsed three times with hexane. The rinse was added to the GC/MS vial. A constant volume, 10uL, of the internal standard, 2, 4-dinitrotoluene-3, 5, 6-d3, was added to each autosampler vial.

Sample Analysis

Samples were analyzed using a Finnigan gas chromatograph with a TRACE TR-5 capillary column (30 m x 0.25 mm i.d.). The Polaris mass spectrometer was run in selected ion monitoring (SIM) mode. The specific ions measured are shown in Table 1. For both the nitromusk and polycyclic musk SIM scan the temperature of the column increased at three different time periods throughout the total run time of 56 minutes. The final temperature for both SIM scan methods was 300 ◦C. The samples were injected manually with a 10 uL chromatography syringe containing 1 uL of air followed by 1 uL of sample and an additional 1 uL of air. Compound Quantification Ion Confirmation Ion

HHCB (galaxolide) 243 258

AHTN (tonalide) 243 258

MX (musk xylene) 282 297

MK (musk ketone) 279 294

Internal Standard 168 92

Table 1: Quantification and Confirmation SIM Scan Ions.

A standard calibration curve was prepared for the four musk compounds and used for

peak identification and quantification. This was done by diluting standards of each of the musk compounds using volumetric flasks, running them on the GC/MS, and calculating the response factor. The response factor for each fragrance compound was calculated as the ratio of the peak area of the compound to a peak area of the concentrated internal standard, ~50 mg/L 2,4-dinitrotoluene-3,5,6-d3. All peak areas were measured from the GC/MS chromatogram quantification ion. The use of the internal standard area was necessary because it accounts for fluctuations in the GC/MS. The response factor was plotted against the dilution concentration to determine a linear trendline.

     

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RESULTS Calibration Curves

Figures 3 and 4 below show the calibration curves created for nitromusks and polycyclic musks, respectively. These calibration curves were created as described above in the previous section. The slope and intercept of the trendline were used to calculate the unknown fragrance chemical concentrations (mg/L) in the MDL autosampler vials as well as the percent recoveries for each musk fragrance chemical.

Figure 3: Nitromusk calibration curve The equation of the trendline is used to determine the unknown concentrations of Nitromusk fragrance chemicals. The R2 values for both trendline equations were less than 0.98, suggesting that human error or instrument sensitivity can be improved upon.

     

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Figure 4: Polycyclic musk calibration curve The equation of the trendline was used to determine unknown concentrations of HHCB and AHTN. The R2 values for HHCB and AHTN were ~0.92 and ~0.93 respectively, suggesting that human error or instrument sensitivity can be improved upon. Method Detection Limits

Method Detection Limits (MDL’s) were calculated for both the nitromusk and polycyclic musk fragrance chemicals in order to determine the lowest measureable concentration for each specific musk fragrance compound. The results can be seen in Figure 5. Aqueous samples spiked with initial concentrations of 2.1, 2.15 0.5, and 0.5 mg/L of MK, MX, HHCB, and AHTN respectively along with two blanks, containing no musk chemicals, were tested following the procedures described previously in the methods section. Eight total samples were run in order to obtain more accurate and reproducible results. The MDL for the nitromusk fragrance chemical MK was significantly higher than that of the polycyclic musk fragrance chemicals HHCB and AHTN. Therefore, the polycyclic musk compounds can be studied at much lower concentrations than the nitromusk compounds. The MDL of MK was 0.11 ug/L and the MDL of HHCB and AHTN was 0.019 and 0.022 ug/L. This may be due to the high sensitivity of the GC/MS to polycyclic musk fragrances, allowing a lower concentration to be detected.

     

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Figure 5: Method Detection Limit Concentrations The data shows the lowest detectable concentration of MK, HHCB, and AHTN in aqueous samples that can be reported with a 99% confidence. Percent Recoveries

The unknown concentrations of the eight samples for each of the four fragrance chemicals were found using the above calibration curves. The initial concentration for MK was 2.10 mg/L and the initial concentration for MX was 2.15 mg/L. The measured concentrations of MK ranged from 1.56 to 2.02 mg/L, with a mean of 1.74 mg/L. The average concentration of MK was less than the initial concentration; therefore the mean percent yield of MK was less than 100%, or ~83% (see Figure 6). The measured concentrations of MX ranged from 0 to 2.36 with an average of 0.87 mg/L. The mean concentration was significantly lower than MK, although they had similar initial concentrations, because five of the eight samples did not show any measureable MX suggesting that the initial concentration of MX, 2.15 mg/L, was too low to be detected. Therefore, five of the eight samples displayed a MX final concentration of zero. Thus the percent recovery of MX was not reported.

The initial concentrations for HHCB and AHTN were both 0.5 mg/L. The concentration of HHCB in the eight autosampler vials ranged from 0.65 to 0.75 mg/L with a mean of 0.70 mg/L. The concentration of AHTN ranged from 0.63 to 0.74 mg/L with a mean of 0.68 mg/L. The mean concentrations of both polycyclic musk fragrance chemicals were above the initial concentration of 0.50 mg/L; corresponding to a percent recovery above 100%. Specifically, the percent yield for HHCB was 140% and the percent yield for AHTN was 136%. A percent recovery over 100% suggests that contamination or human error is likely to have occurred. It is possible that some remaining chemicals from the glassware used in previous experiments may have contaminated the samples, causing the concentrations to be higher than the original.

     

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Another possibility is that the response of the GC/MS changed between the various runs. This is a more plausible explanation because there was no contamination visible in the two blanks.

Figure 6: Percent Recovery of MDL Samples The data for MK, HHCB, and AHTN is reported to show the relation between nitromusk and polycyclic musk percent recovery. The error bars represent the standard deviation of eight samples. Pike River Samples Two aqueous and two sediment samples were taken from the Pike River on Carthage College campus. Samples taken from the Pike River at Carthage College were not found to contain any fragrance chemical concentrations above the method detection limit. There is no wastewater treatment plant discharge that enters the Pike River, so the results support the original hypothesis that fragrance chemicals would be found near WWTP effluents. Fragrance chemicals may still be present in the Pike River at concentrations below the detection limit. For instance, Simonich et al found concentrations of fragrance materials to be 0.005 to 1.7 ug/L near the effluent of a WWTP in Ohio (2000). The MDLs in Figure 5 may be within the range of Simonich’s study, but there are still lower detectable concentrations according to her experiment. The instruments used throughout this experiment may not have the ability to detect these fragrance chemicals at such low concentrations. DISCUSSION AND CONCLUSIONS

Because the concentrations of fragrance chemicals that were measured were so low, any outside contamination could have altered results. Although the researchers in the laboratory were careful not to use soap with fragrances to wash their hands, precautions were not taken regarding non-scented shampoo or laundry detergent use.

A significant amount of time was spent troubleshooting the GC/MS due to instrument malfunction. Many hexane blanks were run to determine if contamination in solvents, glassware,

     

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or instruments was causing a noisy baseline. It was concluded that the GC/MS was very sensitive to interfering compounds and that some plasticizers have been used on the GC/MS in the past that may have been interfering with data analysis. The GC program also tended to freeze in the middle of scans, so many of the sample runs had to be restarted. The autosampler bent needles and would occasionally not pull up samples, so some of the samples had to be injected manually. FUTURE DIRECTIONS

The concern for percent recoveries above 100% for the polycyclic musk fragrance chemicals should prompt the lab members to take more precautions when performing extraction experiments. In future research, perhaps all members in the laboratory should be careful not to use shampoo, soap, detergents, or lotions with fragrances either inside or outside of the lab to reduce risk of contaminating the samples.

Since the data for MX was undetectable in more than half of the MDL samples, a future goal would be to redo the spike recoveries for the nitromusk fragrance chemicals. None of the nitromusk MDL samples were spilled or significantly altered in any noticeable way; therefore, there is no obvious reason why the concentration of MX was so low. The response factor for MX was the lowest of the four fragrance chemicals, however, so perhaps the peaks were just too small to be discerned from the baseline.

An MDL was found only for aqueous samples, so an MDL for sediment samples would need to be determined as well. The clean sand would be spiked with a known concentration of fragrance chemicals, just as the deionized water was for the aqueous samples, and the sediment extraction procedure would be performed.

Some time was spent researching Triclosan, another synthetic chemical found in soap, whose concentrations may be measured, analyzed, and compared to those of musk fragrance chemicals in the near future. Another factor that could possibly be introduced is the location of samples taken. The future focus of this research will be on Lake Michigan, an unmoving body of water. There exists the possibility of moving samples site to the Mississippi River to study how moving water affects concentrations of chemicals. Other potential sites for sampling may be near Chicago and Milwaukee wastewater treatment plant outfalls. It would be useful to know the concentration variations in the area to determine where fragrance chemicals are accumulating.

If future research gives proof that these fragrance chemicals are indeed harmful, then more effective clean-up methods can be proposed for wastewater treatment plants. Another possibility would be to create more organic, fragrance-free personal care products. Dr. Rubinfeld’s previous research shows high concentrations of fragrance chemicals near the effluent of wastewater treatment plants (2008), so clean-up methods could be established to provide a safer aquatic environment in those areas. ACKNOWLEDGMENTS Kayla Tripp and Chelsea Burns would like to thank the SURE program for providing adequate funding for this research project. They would also like to thank Dr. Rubinfeld for being a helpful mentor and providing proper instruction and background information about her research and environmental science as a whole.

     

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BIBLIOGRAPHY

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