HES Study Number 11936
- 108



### Page **1** of **32**

Study Title: **NON REGULATED STUDY: Development of Collection,**  **Storage and Analysis Procedures for the Quantification of D4, D5, and D6 in Waste Water Treatment Plant Effluent**  **and Surface Water Samples**  HES Study No: 11936
- 108 Test Substances: Octamethylcyclotetrasiloxane (D4) Decamethylcyclopentasiloxane (D5) Dodecamethylcyclohexasiloxane (D6) Study Leader: Scott M. Knoerr Study Personnel: Scott M. Knoerr Todd H. Schramke Sponsor: Centre Européen des Silicones (CES) 

### Avenue van Nieuwenhuyse 4, box 2 B - 1160 Brussels Belgium Testing Facility: Dow Corning Corporation Health and Environmental Sciences (HES) 2200 W. Salzburg Road Auburn, MI 48611 HES Manager Chemistry: Debra A. McNett Study Completion Date: 18April 2013HES Study Number 11936
- 108



### Page **2** of **32 TABLE OF CONTENTS**  TABLE OF CONTENTS ................................ ................................ ................................ ................ 2 ABSTRACT ................................ ................................ ................................ ................................ .... 4 SI GNATURES AND APPRO VAL ................................ ................................ ................................ 5 OBJECTIVE ................................ ................................ ................................ ................................ .... 6 TEST ARTICLE SPECIFI CATIONS ................................ ................................ ............................. 6 a. Reference Article 1a
................................ ................................ ................................ ........... 6 b. Reference Article 1b
................................ ................................ ................................ ........... 6 c. Reference Article 2a
................................ ................................ ................................ ........... 6 d. Reference Article 3a
................................ ................................ ................................ ........... 6 e. Reference Article 3b
................................ ................................ ................................ ........... 7 EXPERIMENTAL ................................ ................................ ................................ .......................... 7 A. Background
................................ ................................ ................................ ........................ 7 B. Method Description
................................ ................................ ................................ ............ 7 C. Quality Control Program
................................ ................................ ................................ .. 10 D. High Organic Content Samples
................................ ................................ ........................ 11 E. Linearity
................................ ................................ ................................ ........................... 11 F. Recovery ................................ ................................ ................................ ........................... 11 G. Stability
................................ ................................ ................................ ............................ 12 H. Ruggedness
................................ ................................ ................................ ....................... 12 I. Detection Limits
................................ ................................ ................................ ............... 13 J. Municipal Waste Water Treatment Plant Samples
................................ ........................... 13 DATA ANALYSIS ................................ ................................ ................................ ....................... 14 A. Calculations
................................ ................................ ................................ ...................... 14 RESULTS AND DISCUSSI ON ................................ ................................ ................................ .... 15 A. Quality Control Program
................................ ................................ ................................ .. 15 B. High Organic Content Samples
................................ ................................ ........................ 16 C. Linearity
................................ ................................ ................................ ........................... 17 D. Recovery ................................ ................................ ................................ ........................... 17 E. Stability
................................ ................................ ................................ ............................ 18 F. Ruggedness
................................ ................................ ................................ ....................... 19 G. Detection Limits
................................ ................................ ................................ ............... 19 H. LDPE Mode of Action
................................ ................................ ................................ ..... 20 I. Municipal Waste Water Treatment Plant Samples
................................ ........................... 20 CONCLUSIONS ................................ ................................ ................................ ........................... 22 REFERENCES ................................ ................................ ................................ .............................. 23 TABLES ................................ ................................ ................................ ................................ ........ 24 Table 1. Quality Control Blank Samples WWTP #1 and #2
................................ .............. 24 Table 2. Quality Control Spike Samples WWTP #1 and #2
................................ ............... 24 Table 3. Effect of Organic Content on LDPE Extraction
................................ ................... 25 Table 4. Extraction of D5 by LDPE in a High Solids Matrix
................................ ............. 25 Table 5. Linearity Range of Solvent Standards
................................ ................................ .. 26 Table 6. Recovery of D4, D5 and D6 from Low and High Solids Matrix with LDPE Method
................................ ................................ ................................ ................................ . 27 Table 7. Stability With and Without LDPE of Effluent Solutions Spiked with ~5 μg/L cVMS ................................ ................................ ................................ ................................ ... 28 Table 8. Comparison of Headspace Storage versus LDPE with Effluents Spiked at ~5 μg/L cVMS ................................ ................................ ................................ ................................ ... 28HES Study Number 11936
- 108



### Page **3** of **32** Table 9. Sample Stability Utilizing a Second Supplier of LDPE Film and L DPE Resin
... 29 Table 10. Assessing the Limit of Detection, Method Detection Limit and Limit of Quantitation for D4, D5, and D6
................................ ................................ ......................... 29 Table 11. Results of LDPE Analysis of WWTP #1 Aqueous Samples
.............................. 30 Table 12. Results of LDPE Analysis of WWTP #2 Aqueous Samples
.............................. 30 Table 13. Results of Biosolid Samples WWTP #1
................................ ............................. 30 Table 14. WWTP #1 Results of Aqueous Samples Analyzed by Direct Solvent Extraction

................................ ................................ ................................ ................................ ............. 30 Table 15. WWTP #1
- Percent Difference Comparing LDPE Method to Direct Solvent Extraction of Aqueous Samples
................................ ................................ ........................... 31 Table 16. WWTP #2 Results of Aqueous Samples Analyzed by Direct Solvent Extraction

................................ ................................ ................................ ................................ ............. 31 Table 17. WWTP #2
- Percent Difference Comparing LDPE Method to Direct Solvent Extraction of Aqueous Samples
................................ ................................ ........................... 31 FI GURES ................................ ................................ ................................ ................................ ...... 32 Figure 1. Stability of cVMS in Samples with LDPE, without LDPE, and Low Headspace 32HES Study Number 11936
- 108



### Page **4** of **32**

**ABSTRACT** Due to the ubiquitous nature of cyclic volatile methyl siloxanes (cVMS) in consumer products such as personal care product and anti
- perspirants, cVMS may enter the waterway s and environment through the wastewater treatment plant. The presence of cVMS in the environment has become of regulatory concern for various governmental agencies in determining their potential for persistence, bioaccumulation and toxicity
. This study

describes the development and validation of a method to quantify the amount of oct ameth yl cyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5), and dodecameth yl cyclohexasiloxane (D6) in wastewater treatment plant influent, effluent and surface waters. Th is method utilizes low density polyeth yl ene (LDPE) to inhibit evaporative loss of cVMS during sampling and transport to the laboratory . The sample is extracted into hexanes containing isotopically labeled cVMS as an internal standard and anal yz ed b y gas chromatograph y mass spectrometry. The benefits of the method are its ease of s ample collection and anal ys is preparation. The method detection limit has been demonstrated to be < 0.2 μg/L . In addition, this method has shown recoveries of ≥ 70% during storage of the water samples for up to 14 day s at room temperature conditions. Lin earity of the method has been established from the limit of quantification up to 600 μg/L
. Additionally , due to the ubiquity of cVMS, this report describes a quality control program that should be employed during sampling to ensure sample integrity via contamination as well as sample loss.HES Study Number 11936
- 108



### Page **5** of **32**

**SIGNATURES AND APPRO VAL**  This report consists of 32 pages with 1 figure, and 17 tables . The undersigned have read and approved this report.

**___________________________ _____________________**  **Scott M. Knoerr Date**  **Study Leader**

**___________________________ _____________________**  **Debra A. McNett Date**  **Manager, Chemistry**HES Study Number 11936
- 108



### Page **6** of **32**

**OBJECTIVE** The objective of this study is to develop an anal yt ical m ethod for determining the collection, storage and analy sis procedures for the quantification of octameth yl cyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5), and dodecameth yl cyclohexasiloxane (D6) in waste water treatment plant (WWTP ) influent, effluent and surface water samples
. **T EST ARTICLE**  **S PECIFICATIONS**  Reference article characterization was done in compliance with the EPA Toxic Substances Control Act (TSCA), Good Laboratory Practice Regulations (40CFR Part 792). a. Reference Article 1 a  Id entification: Octameth yl cyclotetrasiloxane ( D4 )  CAS Number: 556
- 67
- 2  Lot Number: LL084732 (supplied as Dow CorningTM 244 Fluid)  Expiration Date: 28Mar2012  Purity : 99.77 % by GC
- FID b. Reference Article 1b  Id entification: Octameth yl cyclotetrasiloxane ( D4 )  CAS Number: 556
- 67
- 2  Lot Number: 0006542999 (supplied as Dow CorningTM 244 Fluid)  Expiration Date: 02Nov2013  Purity : 99. 84 % by GC
- FID

c. Reference Article 2 a  Id entification: Decamethylcyclopentasiloxane ( D5 )  CAS Number: 541
- 02
- 6  Lot Number: 0000341832 (supplied as Dow CorningTM 1693 Fluid)  Expiration Date: 31Dec2010  Purity : 99.16 % by GC
- FID d. Reference Article 3 a  Id entification: Dodecamethylcyclohexasiloxane ( D6 )  CAS Number: 540
- 97
- 6  Lot Number: 0005578887 (supplied as Dow CorningTM 246 Fluid)  Expiration Date: 05Oct2011  Purity : 97.3 % by GC
- FIDHES Study Number 11936
- 108



### Page **7** of **32**

e. Reference Article 3b  Id entification: Dodecamethylcyclohexasiloxane ( D6 )  CAS Number: 540
- 97
- 6  Lot Number: LL114030 (supplied as Dow CorningTM 246 Fluid)  Expiration Date: 14Dec2014  Purity : 99.6 % by GC
- FID **E XPERIMENTAL**  A. Background Cyclic volatile meth ylsiloxanes (cVMS) are common ingredients in many consumer products, including personal care products such as hair conditioners and anti
- perspirants. The wastewater stream represents a major post
- use disposal route and subsequentl y enters the environment through the waste water treatment plant’s (WWTP) effluent. In order to understand the role of WWTP emissions of cVMS into the environment, it is important to be ab le to accuratel y quantify cVMS in aqueous matrices. The primary

objective of the current study was to develop a method to preserve and accuratel y determine concentrations of cVMS in surface waters and waste water treatment plant effluent in an effort to understand the fate of cVMS in the environment. The studied compounds

– octamethylcyclotetrasiloxane (D4), decameth yl cyclopentasiloxane (D5), and dodecamethylcyclohexasiloxane (D6)
- are volatile, have low solubility in water, and very high Henry ’s law c onstants, making accurate quantification in WWTP effluent and associated surface waters difficult. Additionally , these compounds have relatively high values of log KOC 

### – 4.22, 5.17, and 6.12 1 

### , respectivel y - indicating a strong tendency to partition onto o rganic matter. Methods for collection and anal ys is of aqueous matrices must take into account the dissolved and labile fractions of cVMS that may readil y evaporate, as well as those fractions that are associated with the organic content within the water co lumn. Consequently , methods for collection and anal ys is must incorporate some means to sequester the dissolved and labile fractions, as well as recover the sorbed fraction from the suspended solids. B. Method Description LDPE Preparation The LDPE for this study

was obtained from the unprinted material of Ziploc® storage bags. The method utilizes low density polyethylene film (LDPE) , 2
- 2.5 μm, to act as a sorbent and partial barrier to prevent loss of analy te to theHES Study Number 11936
- 108



### Page **8** of **32** headspace of the sample jar. The film was cut into 1” x 1” squares and washed with hexanes and vortexed for 30 minutes on a multitube vortexer. The hexanes was drained, another aliquot of hexanes was added and vortexed again for 30 minutes. The hexanes were drained again and the squares were allowed to air dry
. Sample Collection The water samples were collected in 4
- oz jar equipped with a poly tetrafluoroeth yl ene (PTFE) lined lid. The jars were prepared b y wrapping PTFE tape around the threads of the jar to further inhibit l osses of volatile material from the samples. Into each of the jars were placed 8 of the LDPE squares. The prepared jars were weighed for later determination of sample weight. In the field, the sample was collected by grabbing a sample using a clean stai nless steel bucket which had been rinsed several times with the intended sample. A larger glass container was then also rinsed in the sample and subsequently

used to collect a sample. An aliquot from the jar was poured directly into the sample jar so that the liquid filled approximately half of the 4 oz sample jar (effectivel y colle ct ing 50
- 60 mL of sample) . Samples, in general , were collected in triplicate. A large aliquot of this sample was also retained in a glass jar whose threads had been wrapped w ith PTFE tape and the lid was lined with PTFE . The amount of sample in the jar provided for limited headspace . The sample jars were taken back to the lab in a cooler containing gel pack s to keep them cool. Once in the lab they were weighed to determine the amount of sample that had been added. Sample Preparation The samples were weighed to 0.1 mg to determine the amount of sample that had been added to the jar. To the sample jars were added 10 mLof internal standard working solution (ISWS), a hexanes solution containing ~20 ng/ g of 13C
- D4, 13C
- D5, and 13C
- D6 as an internal standard. The samples were mixed on a Fis her Multi
- tube vortexer on its maximum setting for 30 minutes. When necessary , the samples were centrifuged for 5 minutes at 2400 rpm ( 1570 relative centrifugal force) to get greater separation between the two phases. An aliquot of this sample extract was removed for anal ys is by gas chromatography

/mass
spectrometry

(GC
- MS)
. Biosolids Sample Preparation The method used for biosolids was described in an earlier report2,3,4



### . Approximately

2.5 g of sample was weighed into a 20
- mLscintillation vial followed b y 2.5 mL of acetonitrile and 5 mL of IS WS . The mixture was vortexed on high for 30 minutes and centrifuged at 2000 rpm (1308 relativ e centrifugal force) for five minutes. The extract was removed and added to another vial. An additional 5 mLof hexanes was added to the sample containing acetonitrile andHES Study Number 11936
- 108



### Page **9** of **32** vortexed and centrifuged as described above. The extract was removed and combined with the original extract and an aliquot was anal yz ed b y GC
- MS . Samples were anal yz ed with triplicate preparations
. Direct Solvent Extraction Sample Preparation From the large aliquot of sample that was retained during collection , 5 0 mL aliquots of sample were weighed into 4
- oz jars along with 10 mL of the IS WS . The mixture s were vortexed on high for 30 minutes and the phases allowed to separate. An aliquot of the organic layer was removed for anal ys is by GC
- MS. Samples were anal yz ed with triplicate preparations
. LDPE Sample Analy sis Samples were separated and quantified on a GCMS utilizing a DB
- Wax etr or DB - 5ms chromatograph y column for separations while the mass selective detector was operated in selected ion mode. Standard solutions were prepared with D4, D5, and D6 in ISWS as internal standards. A calibration curve was constructed comparing the concentration ratio of the test article to the internal standard in the standard solutions against their relative chromatographic response. If sample results exceeded the calibration curve range , they were further diluted with IS WS and reanalyzed . During anal ys is, the samples were bracketed by solvent blanks and standards to ensure the absence of carryover and that the standard curve remained within c ontrol limits
. In order to ensure a valid calibration curve and analy sis, the coefficient of determination, R2 

### , of greater than 0.995 was the criteri on to assess the fit of the linear regression . Additionally , when determining the recovery of the standards against the calibration curve, a relative error of less than 20% was used as the guideline.HES Study Number 11936
- 108



### Page **10** of **32** Chromatographic Conditions Equipment Agilent 7890 GC coupled with 5975 MSD and a CTC Analy

tics PAL autosampler Column DB - WAXetr 30m x 0.25mm , 0.25 μ film DB - 5ms 30m x 0.25mm, 0.25 μ film Injection Size 4 μL Inlet Temperature 150 °C Inlet Pressure (constant) 18 psi Inlet Mode Splitless (5 mL/min at 0.1 min) Oven Temperature 40 °C for 3 min, 40 °C/min to 150 °C, no hold, 60 °C/min to 240, no hold Transfer Line Temperature 260 °C MS Source Temperature 230 °C MS Quad 150 °C Selected Ions (dwell time 20 ms) 281, 285, 355, 360, 429, and 435 C. Quality

Control Program Due to the ubiquity

and volatile nature of cVMS, it is important to implement a quality control (QC) program to ensure the integrity of the samples and analy sis from the point of sample collection to anal ys is. Potential problems faced when anal yz ing for trace levels of cVMS in water samples are contamination from the environment, analy st, equipment, and tools as well as sample loss during collection, transfer, and sample preparation. In order to mitigate these problems, a quality control program demonstrates that samples were handled and stored in a way to minimize contamination as w ell as analy te loss. For these studies, the quality control samples utilized were trip blanks, field blanks, and field spikes. The spikes and blanks were all prepared similarly in that they were 4 oz jars whose threads were wrapped with PTFE tape and cap ped with a PTFE lined lid. The jars each had 8 squares of LDPE and contained approximately 50 mL of sample. Each QC sample was prepared in triplicate. The trip blank consisted of milli Q water and was prepared at the onset of a sampling trip. It remai ned sealed until the time of sample preparation. This sample was used to demonstrate contamination free glassware and test materials and/ or proper sealing of the jars to prevent contamination. The field blank also consisted of milli Q water but was open ed during the time of sampling to determine if there was ambient c ontamination that could possibly deposit into the samples at that time. These blanks were closed after sampling and anal yz ed with the collected samples.HES Study Number 11936
- 108



### Page **11** of **32** The field spike was prepared at the sampling site by taking an aliquot of the sample (effluent or influent) and placing into the prepared jar. The sample was then spiked with 25 μLof D4, D5, and D6 at a relevant concentration for the sample ( e.g. effluent was spiked at approximately 4 μg/ L and influent at 3800 μg/Lfor site #1 and 1 1 0 μg/L for site #2 ). This spike ’s purpose was to show that the anal yt e was sufficiently retained by

the sample container

/ LDPE and no degradation of the anal yt es occurred due to the matrix or the collection conditions. D. High Organic Content Samples R elatively

high log Koc and Kaw 5 

### values are found for cVMS, indicating that they readil y partition to suspended matter/organic content in the water column as well as evaporate from the aqueous sample. The efflu ent contained low suspended solids, ~4 mg/L , and therefore low organic content. Post digested biosolids were added to the effluent to affect a higher organic content , boosting the solids content to ~42 mg/L
. To determine the magnitude of this effect on the developed method, effluent and the amended effluent were spiked in bulk with approximately

4
- 6 μg/LcVMS. Aliquots of this bulk material were extracted onto the LDPE, the LDPE removed and both the remaining aqueous phase and the LDPE extracted with 10 mLof the hexanes based internal standard solution. As a result, the calculation of a mass balance of total cVMS and how much partitioned to the LDPE versus that which stayed in the aqueous and suspended solids phase could be determined
. The impact of ti me the samples were vortexed with LDPE was also evaluated. E. Linearity The calibration curve constructed with the standard solutions was used to establish the linearity

of this method. F. Recovery The recovery

of cVMS from the samples was studied by spiking effluent samples with D4 , D5, and D6 in tetrah ydrofuran from ~0.5 to 1100 μg/Lmaking sure to place the spikes directl y beneath the surface of the liquid. Each sample was spiked individually , rather than spiking in bulk and then aliquoting
. This addressed concerns regarding the ability of the matrix and sample container to prevent evaporation of cVMS , as well as whether the cVMS would be homogeneously

distributed throughout the matrix. The effluent used as the sample matrix had a suspended solids content of 12 mg/L , which can be considered a relativel y low amount as 10

– 30 mg/L is ty pical 6 

### . Since the suspended solids are low that means the organic content is alsoHES Study Number 11936
- 108



### Page **12** of **32** relativel y low. In order to demonstrate that the method can also recover cVMS from samples containing higher amounts of organic content, effluent sample was amended with post
- digested biosolids to reach % solids content of 47 mg/L
. G. Stability The stability

samples were prepared as described for the recovery samples except that the targeted concentration of the cVMS in the samples was 5 μg/L. Stability of the cVMS in the effluent with LDPE was assessed at 5 °C and at ambient temperature over the course of 14 days . To determine if headspace had an impact on the ability

of this testing sy stem to retain the cVMS, a series of samples were also tested for stability where 2 oz jars were used in place of the normal 4 oz jar. The sample only occupies approximately

1⁄2 o f the 4 oz jar jar, while the sample consumes nearl y the entire volume of the 2 oz jar
. Additionally , samples were prepared that contained no LDPE to act as control to demonstrate the storage loss. The samples from this portion of the study were processe d somewhat differently than the methodology

described above. At each predetermined time point the samples were vortexed for 30 minutes and then the LDPE was removed from the sample and placed in a 20
- mLscintillation vial for extraction into the hexanes b ased internal standard solution. The vials were then vortexed for 30 minutes. Samples that were not stored with LDPE were processed b y liquid
- liquid extraction into 10 mLof the hexanes based internal standard solution and vortexed 30 minutes. Another f orm of collection noted in literature is the use of 100
- mLheadspace vials with sample work up using a membrane assisted solvent extraction sy stem 7



### . As part of this stability study , effluent was added to the 100
- mLheadspace vials and then spiked at ~5 μg/L with cVMS. The referenced method fills the vial full and then caps the vial leaving no headspace. In this spiking experiment headspace was left to ensure the spiked material was not lost
. H. Ruggedness A second supplier of the LDPE film (Griff Paper an d Film, 2 mil LDPE Clear
Base film) was used to ascertain the ruggedness of this methodology . Due to the amount of pre
- work required cutting the LDPE films into the 1 x 1 inch squares , LDPE resin (Dow Chemical LDPE 5004I) was evaluated to see if it would be a suitable replacement for the film. The resin utilized 20 pellets in e ach sample. Each pellet was ellipsoid in shape with an approximate surface area of 49.3 mm 2



### .HES Study Number 11936
- 108



### Page **13** of **32** The detection limits as well as sample stability were evaluated using the different supplier of LDPE and the resin. The samples were prepared as described in the Sample Preparation section. I. Detection Limits The detection and quantitation limits of the method were evaluated several times during the course of this method development and sample anal ys is to assess the suitability of the method and equipment at the time of anal ys is for samples. The limit of detection (LOD) was defined as the minimum level of constituent that can be detected and reported with 99.9% probability that is gre ater than a matrix
- free blank and was calculated as 2 x tn - 1, α1=0.05 

### x s where s is the standard deviation of seven injections of hexanes based internal standard solutions 8,9



### . The method detection limit (MDL) was defined as the level of analy te in a sample matrix, that when processed through the complete anal yt ical method, can be measured and reported with 99% probability that it is greater than zero and was calculated as t

n - 1, α1=0.01 

### x s wh ere s is standard deviation of seven replicate preparations and anal ys is of effluent. The limit of quantitation of a method was defined as the minimum level of anal yt e in a sample matrix that can be measured and accuratel y quantified with an estimation error no greater than 30% with 99% probability which was calculated as 2.5 times the MDL
. J. Municipal Waste Water Treatment Plant Samples Two municipal waste water treatment plants (WWTP) were targeted for collection of samples and analy sis of the various streams for cVMS content with the purpose of verifying that the methodology described in this study performs well. The studied WWTPs operate under the same principal where the influent p asses through a degritter to remove large debris. The influent is discharged into settling tanks for the primary

treatment, where solids are settled out and floating material is skimmed from the surface. The primary effluent then undergoes secondary treatment in an aerated basin with activated sludge to further breakdown the organic material. The activated sludge is forwarded to the final clarifiers allowing the solids to drop out and the effluent to be either discharged or undergo chlorine treatment pri or to discharge. At WWTP # 1 , multiple streams and solid samples were sampled throughout the treatment process. Additionally , air monitoring was conducted at WWTP # 1 . At WWTP #2 only the influent and effluent were collected and anal yz ed.HES Study Number 11936
- 108



### Page **14** of **32**

**D ATA ANALYSIS**  A. Calculations In calculating the amounts of test article present in the various samples and matrices, various corrections were performed to account for matrix and analy tical effects . The values from samples collected from the WWTP sites were corrected usi ng the values from internal standard blanks. Validation and developmental samples were corrected for matrix blanks , either effluent or milli
- Q water
whichever was appropriate. The spikes of the various WWTP samples, performed as part of the quality control program, were also corrected for the loading of test article native to the matrix. Additionally the raw area counts were corrected to account for ion overlap between the parent ions of the test articles and their respective isotopically labeled count erparts that were used as internal standards. Exponential regression equation of the line and equation for sample analy sis

= + and = ( ) 

### Where: y = Area response ratio of test article and the internal standard m = slope of the line x = Concentration ratio of test article and the internal standard b = y
- intercept Test Article (TA)

= ( ) Where: TA = Amount of test article , ng x = Concentration ratio of test article and the internal standard ISTD = Amount of isotopically labeled test article added, ng Percent Relative Error (%RE) % = [ ] 

### 100 Where: %RE = Percent relative error, % F = Found test article , ng T = Theoretical test article , ngHES Study Number 11936
- 108



### Page **15** of **32** Percent Recovery

(%Rec) % = 100 Where: %Rec = Percent recovery, % F = Found test article , ng T = Theoretical test article , ng **RESULTS AND**  **D ISCUSSION**  A. Quality

Control Program The QC program as described above was executed at both WWTP collection sites. All QC samples were pr epared and anal yz ed in triplicate. At both sites the recovery of the field spikes ranged from 81 .6

– 107.3 % with the %RSD less than 4.9 % in all but one case, and that value was 13.3%
. The QC sample results are shown in Table 1. This demonstrated that the sample jars, as prepared, along with LDPE were successful in preventing significant evaporative losses and degradation of D4, D5, and D6. The blanks prepared and anal yz ed for WWTP #2 were all below the defined L OD as shown in Table 2 for th e respective analy tes except one value that was between th e MDL and the LOD. The largest LOD for all anal yt es was 0. 094 μg/L
. The MDL for one analy te is 0. 170 μg/L . This demonstrated that the blanks were not significantly contaminated due to the sample jar preparation, ambient conditions during sample collection at the site, storage, transfer or sample preparation/extractions. The Trip blank for WWTP #1 was below the LOQ in all cases, but the influent and effluent field blank s all had quantifiable amo unts for D4 and D5. There was significant variation between the 3 samples for both influent and effluent which at the measured level is unusual. In the case of the effluent, the amount actually measured in the effluent samples is lower than the amount f ound in the effluent field blanks indicating that whatever affected the blanks did not affect the effluent samples themselves. For the influent, the %RSD of the blanks was significantly higher, greater than tenfold, than that measured for the actual influent samples. The average of the blank was less than the samples for D5 and D6 but higher for D4. It is suspected that the blanks were contaminated in a way that did not affect the samples; however, there was no direct proof of this. A corrective action taken due to the spurious blank results was that the field blanks were onl y open during the sample collection and not during the spiking of the field spikes.HES Study Number 11936
- 108



### Page **16** of **32**

B. High Organic Content Samples Two different matrix sets were anal yz ed with differing amounts o f solids and organic content. The effluent contained ~4 mg/L suspended solids and the amended effluent had ~42 mg/L solids and both were spiked in bulk with 4
- 6 μg/LcVMS. All samples were prepared and analyzed in triplicate. The samples were anal yz ed s uch that the amount of cVMS that was extracted by the LDPE and the amount that remained in the aqueous phase could be measured independently to determine an extraction efficiency of the LDPE with 30 minutes of vortexing. A set of samples was also anal yz ed where the LDPE and aqueous phase were extracted together. The data shown in Table 3 clearl y shows the amount of solids/organic carbon has an effect on how well the LDPE can extract cVMS from the matrix , but when extracting both the LDPE and the aqueous phase, the majority , at least 7 5 %, of the cVMS is recovered from the effluent except for D4 at 48% . It is suspected that the lower recovery

values for the effluent results are an artifact of how the effluent is spiked in bulk and then allowed to acclimate for 24 hours prior to aliquots being taken. Due to the high Kaw of D4 , 2.69 5 

### , it is most likely lo st due to evaporation. The data show that D6 has a greater affinity for the aqueous phase that contains high solids content than does D5 , and D5 more than D4. This is made evident b y the percent extraction efficiency noted in Table 3. T he lower extracti on efficiency

means more of the compound was retained by the aqueous phase. For the effluent, the percent extraction efficiencies were 98.3, 82.7, and 65.5 for D4, D5, and D6, respectivel y. In the amended effluent the results were 88.4, 50.6, and 43.1. These results show that the higher molecular weight species either partition in a greater proportion and/or bind more tightl y to the solids than the lower molecular weight species. This is consistent with their reported log Koc values of 4.22, 5.17 , and 6 .12 for D4, D5, and D6 1



### . Also noteworthy is that the total recovery

for the samples where the LDPE and remaining aqueous phase were extracted separatel y into hexanes is very similar to the samples wh ere the LDPE and aqueous phase were extracted together i nto hexanes , indicating either extraction technique works well. In another experiment , effluent was amended with post digested solids to understand whether further extraction time allows for full recovery of the test article onto LDPE. With the addition of the post
- digested solids, the solids content was ~50 mg/L. In this experiment, no test article was spiked and only native material , D5, was used to assess this effect. As seen in Table 4 , the samples were vortexed with LDPE for 30 minutes and up to 24 0 minutes. The LDPE was removed and extracted into hexanes containing internal standard. The aqueous layer was also analyzed. The values shown are the percent of D5 extracted by

LDPE against the total recovered from both the LDPE and the aqueous phase. The amount extra cted by the LDPE increased over time starting at 24.6% at 30 minutes to 85.8% after 240 minutes of vortexing. ThisHES Study Number 11936
- 108



### Page **17** of **32** demonstrates that the D5 does preferentiall y partition from the organic carbon to the LDPE, but the process requires time and energy
. It also show s the potential for LDPE to be used as a means to measure the freel y labile portion of D5 in these samples, but would require further study. C. Linearity Linearity of this methodology was established b y using the prepared solvent s tandards. Linearity , as shown in Table 5, of the method was demonstrated from ~0.6

– 1200 μg/kg in solvent (equivalent to ~0.08

– 150 μg/L in the samples) with a coefficient of determination of 1.000 for each of the compounds. The calibration curves were e stablished with a linear regression of the concentration ratio of test article to internal standard on the x
- axis and the area response ratio on the y
- axis. The percent relative error was determined by using the area response ratio of each standard to cal culate the amount of test article in the prepared standard. The difference between the found and theoretical (or prepared) amount of test article was divided by the theoretical (or prepared) and then multiplied by 100. The highest percent difference foun d was 3.9%. D. Recovery Low Solids The recovery

of the method was demonstrated in an effluent matrix containing ~ 5 mg/ L suspended solids with the spiked concentrations encompassing 0.493

– 594 μg/Lfor D4, 0.512

– 1232 μg/L for D5, and 0.454

– 1094 μg/L for D6. The recoveries ranged from 63

– 83% of the spiked amount for all test articles. As shown in Table 6, the upper spike for D4, 1189 μg/L, had a poor recovery for undetermined reasons. High Solids The re covery

of this method was also demonstrated on an effluent matrix which had been amended with post
- digested bios o lids to a solids content of ~47 mg/L
. As a resul t of the post
- digested biosolids, the amended effluent contained a background level of cVMS that interfered with the ability of the recovery study

to determine a robust range for D5. The D5 content of the amended effluent was 1.5 μg/L , essentiall y limitin g the lower range of the recovery study to this level. The other cVMS, D4 and D6 were not similarl y impacted due to the low native levels present. The demonstrated ranges, as show in Table 6, were 0.493

– 594 μg/L for D4, 5.95

– 1232 for D5, and 0.454

– 1094 for D6. The recoveries at these ranges were 60
- 87%. As was noted in the low solids recovery study , the highest studied level for D4 had poor recovery for undetermined reasons.HES Study Number 11936
- 108



### Page **18** of **32**

E. Stability The ability

of the LDPE and closure s ys tem to sequester ~5 μg/LcVMS was demonstrated for 14 day s at both ambient temperatures and at 5 °C. It should be noted that since these samples were processed differentl y and the LDPE was separately extracted into the internal standard solution , these results show that the LDPE is the keeper of the anal yt es in this low suspended solids effluent matrix. The stability

results can be seen graphicall y in Figure 1 and tabulated in Table 7. In both the tabular and graphical representations of the results, the data contains samples with LDPE, without LDPE (Control), LDPE with low headsp ace (LHS), and without LDPE LHS. Inall cases, there was not a significant difference between the regular LDPE samples and those samples with LDPE and a low headspace. Generally , the control samples were higher than the control samples with low headspace. A dramatic increase in the stability of the samples containing LDPE was seen immediately for D4 with recoveries for the samples achieving ≥77% vs the samples containing no LDPE , whose recove ries were all ≤ 27% . An advantage was also noted for D5 where the minimum recovery over 14 day s was 76%. For D6 at 5 °C, all samples were above 62% recovery at each time point for 14 day s . The samples containing LDPE showed recoveries of 65 % and above. At ambient temperature for D6, the LDPE samples and the control sample began to deviate starting at day 7 with the day

14 time point being 51% recovered. The control with low headspace began deviating at day 4. It is h ypothesized that the reason for the low headspace control samples being consistently

lower than the control samples may be due to the PTFE lids acting as a sorbent and retaining some of the cVMS. The samples were extracted in another jar so this fr action that may have sorbed to the lid would not have been accounted for. This hy

pothesis was not evaluated in the laboratory
. The data for the 100
- mLheadspace vial experiment is shown in Table 8. The headspace storage method had considerable loss of D 4 after 1 day of storage at 5 °C with a recovery

of only 59%. The LDPE method performed better than the headspace storage method for D5; however, the headspace storage recovery was accep table at 76% after 1 day of storage and 67% after 4 days. The headsp ace storage method seemed to perform better than the LDPE method with recoveries of 94% and 82% for day

1 and day

4 , respectivel y , while the LDPE method dipped to 67% recovery
. Due to the 40% loss in 24 hours o f the spiked D4 seen in this experiment, the headspace vial collection method may be inappropriate or at least deserves further evaluation. Although the above data clearl y shows that the LDPE film helps prevent significant volatilization in samples that are spiked with cVMS , future experiments may

want to evaluate s ys tems that are in equilibrium as these may have a longer shelf life than do samples whose equilibrium are perturbed byHES Study Number 11936
- 108



### Page **19** of **32** spiking additional cVMS. This perturbation may cause loss of the unbound cVMS while the sampl e seeks to reach a state of equilibrium between the labile and bound cVMS. F. Ruggedness LDPE from a second supplier was used to determine sample stability of samples spiked with ~4 μg/L cVMS as described above including ambient and 5 °C temperature conditi ons. The time points prepared were 0 day , 7 day s, and 14 day s. The sample extractions were executed as described in the Sample Preparation section. As shown in Table 9 the second supplier LDPE film performed well at 5 °C with recoveries over the range of 81

– 91 % for D4, D5 and D6 after 14 day s of storage . At ambient temperature the film did not perform as well for D4 through 14 day s as shown by

the 58 % recovery , but D5 and D6 results were acceptable at 83 and 86% recovery , respectivel y. However, af ter 7 day s the D4 results at ambient temperature was acceptable with 71% re covery . The second source of the film performed well for all three anal yt es after 7 day s of storage at ambient temperature. Under refrigerated temperature, 5 °C, the film showed g ood sample integrity over 14 days. A similar set of samples was prepared with effluent samples containing 20 pellets of the LDPE resin and analyzed in a similar fashion. At ambient temperatures the resin did not adequatel y help the stability of the sampl e as the recoveries were 28

– 60% after 7 day s of storage. The recoveries were worse for 14 day s with 24

– 43%. At 5 °C, the samples fared slightl y better but not sufficient to use as a means for storage. After 7 day s of storage , the resin demonstrated 47
- 91% recovery and after 14 days exhibited 49
- 81 % recovery . In both the ambient and the refrigerated experiment, D4 was retained the least. G. Detection Limits The limit of detection, method detection limit and limit of quantitation were evaluated four t imes during this study and the results are tabulated in Table 10 . The LOD was anal yz ed using 7 replicates of LDPE extractions into the I SWS. The MDL and LOQ were anal yz ed from 7 replicate samples of effluent with the exception of the MDLand L OQ for D5 f rom WWTP #2 which used 6 replicates due to an outlier result 10



### . Insufficient effluent was collected from WWTP #1 so the MDLand LOQ for this data set collected was determined using Milli
- Q water. It was noted that there was some variability in the limits found between the sets of samples so it is important that the limit of detection, method detection limit and the limit of quantitation be evaluated with each set up samples using the same LDPE stoc k as used in the samples to ensure an accurate comparison. In one case the LOD for D4 was significantl y greater than the MDL for undeterminedHES Study Number 11936
- 108



### Page **20** of **32** reasons and is not consistent with the other results for LOD. This result is reported in the T able 10 and used in calculating the average LOD. Using the averages, the limits of detection for D4, D5, and D6 were 0.0 4 1 , 0.0 41 , and 0.0 2 5 μg/L, respectively . Similarly , the me thod detection limits were 0.0 37 , 0.1 23 , and 0.0 36 μg/L with a limit of quantitation of 0.0 9 3 , 0.3 07 , and 0. 091 μg/L , respectivel y. H. LDPE Mode of Action The mode of action for the storage of cVMS b y the LDPE was not investigated, however it is suspected that the cVMS adsorb to the surface of the LDPE rather than penetrating the LDPE and absorbing. If adsorbing is the primary means of sequestration, then the most important parameter to prevent volatilization is the surface area of the LDPE relative to the sam ple and or the sample surface. The surface area of the LDPE film is 10323 mm2 

### accounting for the two primary surfaces of all eight square s, with each square having a surface area of 645 mm 2 

### on each face. The weight of all 8 LDPE squares was 0.24 g. The surface area of the LDPE resin was calculated as being 987 mm 2 

### for all 20 pellets with each pellet having a surface area of 49 mm2 

### . The weight of all 20 LDPE resin pellets was 0.68 g. The sample size is 50 mLand in the 4 oz jars utilized, the surface area of the exposed sample is 128 mm 2



### . As shown above the surface area of the LDPE film is far greater than the surface area of the sample; however the film rarel y laid flat and often curled up in the sample. Given the disparity between the surface area of the film and the resin pellets, a more relevant experiment would be to add nearl y 200 pellets to get nearl y the same surface area, however this may present other sample preparation and extraction problems because this would also mean the amount of pelle ts would be 6.8g, potentially interfering with extraction mixing. It is not uncommon for WWTP to collect samples using plastic containers and tubing. When anal yz ing for cVMS, the collection method should be scrutinized to ensure that the cVMS is not lost through adsorption/absorption to the equipment used. I. Municipal Waste Water Treatment Plant Samples Air Monitoring Air monitoring was performed at WWTP #1 at the influent, aeration basin, and the effluent sampling locations. The samples were collected on carbon collection tubes at a sampling rate of ~1.1 L/min and were collected for ~90 minutes. Only D4 and D5 were anal yz ed and both were below the reporting limit of 10 μg at all three locations.HES Study Number 11936
- 108



### Page **21** of **32** LDPE Results

– Municipality #1 Samples were collecte d from the influent , primary settling tank, aeration basin, secondary clarifier, and effluent for anal ys is by the LDPE method outlined in this report. All samples were collected in an afternoon so no conclusions can be drawn about the removal efficiency o f the WWTP process as the h ydraulic retention time of each portion of the process was not considered. The results from the anal ys is by the LDPE method can be found in Table 1 1 . The results shown have been corrected for LDPE blanks. In all locations, D5 was measured in greater quantity than D4 and D6 . The aeration basin contained the highest levels of all the anal yt es than the other testing locations with D4 present at 0.572 μg/L , D5 25.828 μg/L, and D6 5.492 μg/L . The influent contained 0.178, 6.738, and 1.225 μg/L of D4, D5, and D6, respectivel y, whereas the effluent levels of D4 and D6 were below the method detection limits of 0.019 and 0.044 μg/L and D5 was found at 0.292 μg/L. LDPE Results

– Municipality #2 Samples were collected from the influent and the effluent for anal ys is. All samples were collected in an afternoon and as in WWTP #1, no conclusions can be drawn about removal efficiency . The results from analy sis by the LDPE method are shown in Table 1 2 . The results in the table are correc ted for LDPE blanks. The influent samples were found to contain 0.176 μg/L , 8.150 μg/L, and 1.928 μg/Lof D4, D5 and D6, respectivel y, and the ef fluent levels were below the method detection limits of 0.032 and 0.070 μg/L for D4 and D6. D5 was 0.179 μg/L
. Biosolids

– Municipality

# 1 Solids were collected from the digester, primary clarifier, dissolved air floatation (DAF) unit, final clarifier, and buoyant material of the primary clarifier. The DAF unit is an extension of the aeration basin such that it removes a portion of the solids in the basin to help maintain a healthy population of micro
- organisms. The above samples were anal yz ed in triplicate using the biosolids method described above
. As noted in Table 1 3 , a significant portion of cyclics (relative to the water m atrices) are found in the solid samples, the majority of which is D5 and ranged from 210.8 μg/kg in the sludge from the final clarifier up to 1369.1 μg/kg in the sludge from the DAF. The highest measured amount of D4 was 35.0 μg/kg , which was from the sludge from the primary clarifier and 317.4 μg/kg of D6 which was found in the solids from the DAF.HES Study Number 11936
- 108



### Page **22** of **32** Direct Solvent Extraction of Aqueous Samples

– Municipality # 1 The results from the direct solvent extraction of the aqueous samples are displayed in Table 1 4 . This method ology was not validated and a method detection limit was not estimated, but rather used as a comparative anal ys is. The comparison of the direct solvent extra ction to the LDPE method is shown in Table 1 5 . When comparing the two methodologies the values were all below 16 % relative difference for D4, D5, and D6 in all of the results that were greater than 0.5 μg/L b y the LDPE method. For results below 0.5 μg/L , the % difference value was greater and assumed to be due at least in part to approaching the LOQ of the LDPE metho d. Direct Solvent Extraction

– Municipality #2 The direct solvent extraction results and comparison of the samples from WWTP#2 are shown in Tables 1 6 and 1 7 , respectively . Similar to the results from WWTP#1, all resu lts above 0.5 μg/L were below 22 % relative difference. **C ONCLUSIONS**  A method was developed for the collection and storage of WWTP influent, effluent and surface water samples as well for the quantitation of cVMS in these samples. The methodology requires the use of quality control samples in the form of trip blanks, field blanks, and field spikes to demonstrate sample integrity throughout the process. The stability of th e sample was demonstrated for 14 day s at 5 °C using both sources of LDPE film. T he stability was demonstrated for 7 day s at ambient laboratory conditions. LDPE resin pellets did not sufficiently retain the spiked cVMS. The anal yt ical method was demonstrated over the range 0.493

– 594 μg/L for D4, 0.512

– 1232 μg/L for D5, and 0.454

– 1094 μg/L for D6 in samples containing both low and high suspended solids
. The average method detection limit s for D4, D5, and D6 were 0.037, 0.123, and 0.036 μg/L, respectively
. Samples containing a higher amount of solids a ffected the ability of the cVMS to adsorb to the LDPE which is why both the LDPE and aqueous portion of the sample must be extracted to get a total cVMS value.HES Study Number 11936
- 108



### Page **23** of **32**

**REFERENCES** 1. Kozerski, G.E.;Evaluation o f Models for the Estimation of Soil/Sediment
- Water Distribution Coefficients of Cyclic volatile Methylsiloxanes, Dow Corning Corporation, Auburn, MI, 2008. Report to Silicones Environmental, Health, and Safety Council: Study #10847
- 102. 2. Knoerr, S.M.; Durham, J.A.; McNett, D.A. Non
-regulated Study: Development of a Method for the collection, Storage, and Analysis of Biosolids Obtained from a Waste Water Treatment Plant for the Quantitation of Dodecamethylcyclohexasiloxane (D6). Dow Corning Corporation , Auburn, MI, 2012. Report to Centre Europeen Des Silicones: Study 11800
- 108. 3. Knoerr, S.M.; Durham, J.A.; McNett, D.A. Non
-regulated Study: Development of a Method for the Collection, Storage, and Analysis of Biosolids Obtained from a Waste Water Treatme nt Plant for the Quantitation of Decamethylcyclopentasiloxane (D5). Dow Corning Corporation, Auburn, MI,2012. Report to Centre Europeen Des Silicones: Study 11800
- 108. 4. Knoerr, S.M.; Durham, J.A.; McNett, D.A. Non
-regulated Study: Development of a Method for the Collection, Storage, and Analysis of Biosolids Obtained from a Waste Water Treatment Plant for the Quantitation of Octamethylcyclotetrasiloxane (D4). Dow Corning Corporation, Auburn, MI, 2012. Report to Centre Europeen Des Silicones: Study 11800
- 108. 5. Xu, S.; Kropscott, B. M ethod for Simultaneous Determination of Partition Coefficients for Cyclic Volatile Methylsiloxanes and Dimethylsilanediol, Anal. Chem. , 2012 , 84 (4), pp 1948

– 1955. 6. Grady, C.P.L.;Daigger, G.T.; Lim H.C. Biological Wastewater Treatment

– Second Edition, Revised and Expanded; Marcel
- Dekker: New York, 1998
. 7. De - Gao Wang, Helena Steer, Tara Tait, Zackery Williams, Grazina Pacepavicius, Teresa Young, Timothy Ng, Shirley Anne Smyth, Laura Kinsman, Mehran Alaee, Concentrations of cycl ic volatile methylsiloxanes in biosolid amended soil, influent, effluent, receiving water, and sediment of wastewater treatment plants in Canada, Chemosphere, Available online 21 November 2012, ISSN 0045
- 6535, 10.1016/j.chemosphere.2012.10.047. 8. Taylor, J.K . Quality Assurance of Chemical Measurements; Lewis Publishers: 1987. 9. Eaton, A.D.; Clesceri, L.S.; Rice, E.W. Standard Methods for the Examination of Water & Wastewater: Centennial Edition; Amer Public H ealth Assn: 2005
. 10. Barnett, V.; Lewis, T. Outliers in Statistical Data (3 rd edition), Wiley 1994.HES Study Number 11936
- 108



### Page **24** of **32**

**TABLES** Table 1. Quality Control Blank Sample s WWTP #1 and #2

* MDL determined with n=6. 

### Table 2. Quality Control Spike Samples WWTP #1 and #2 D4 , μg /L (% RSD ) D5 , μg

/L (% RS D) D6 , μg

/L (% RS D) WWTP #1 Tr ipBl

an k <MD L <L OQ <MD L Infl ue nt Bl an k 0. 328 (9 7. 0) 0. 223 (4 5. 8) <LO Q Effl ue nt Bl an k 1. 076 (1 40. 8) 0. 397 (1 04. 8) <LO Q MDL 0. 019 0.083 0.044 LOQ 0. 047 0.207 0.111 WWTP #2 Tr ipBl

an k <LO D <L OD <LO D Infl ue nt Bl an k <LO D <L OD <LO D Effl ue nt Bl an k <LO D <MD L <LO D LOD 0. 040 0.094 0.047 MDL 0. 032 0. 170* 0.070

D4 , % Recove ry (% RS D) D5 , Re cove ry

(% RSD ) D6 , Recove ry

(% RS D) WWTP #1 Infl

ue nt Fi el d Spi ke 83. 3 (4 .0) 90. 0 (3 .1 ) 91. 9 (1 .0) Effl ue nt F ie ld S pi ke 82. 7 (0 .5 ) 81. 6 (0 .9 ) 81. 8 (1 .4) Hi gh Spi ke C he ck 85. 7 (3 .1 ) 90. 6 (1 .8 ) 91. 7 (0 .9) Lowsp ike C he ck 86. 8 (2 .4) 90. 1 (0 .8 ) 86. 2 (4 .9) High /I nflue nt S

pike ~3 800 μ g/L Lo w/Efflue nt S

pi ke ~3 .6 μg /L WWTP #2 Infl

ue nt Fi el d Spi ke 87. 6 (1 .0) 97. 0 (1 .3 ) 92. 7 (1 .1 ) Effl ue nt F ie ld S pi ke 87. 3 (1 .6 ) 94. 6 (0 .9 ) 90. 5 (0 .6) Hi gh Spi ke C he ck 98. 8 (0 .3 ) 97. 5 (0 .3 ) 97. 7 (0 .1 ) Lowsp ike C he ck 98. 9 (0 .8 ) 107. 2 (1 3. 3) 99. 2 (1 .3 ) High /I nflue nt S

pike ~1 10 μg /L Lo w/Efflue nt S

pi ke ~4 .3 μg /LHES Study Number 11936
- 108



### Page **25** of **32**

Table 3. Effect of Organic Content on LDPE Extraction

% Extraction Efficiency = Amount Found on LDPE/(Amount Found on LDPE + Amount Found in Water After LDPE Extraction) x100 %Recovery = Amount found/Amount Spiked x 100 

### Table 4 . Extraction of D5 by LDPE in a High Solids Matrix

LDP E Ex tra cti on Wa te r After LDP E

Extr action Tota l Re cove red Combi ne d Ex tra cti on D4 Effl

ue nt % R ecove re d (% RSD) 47. 5 (3. 2) 0. 8 (1 6) 48. 3 47. 7 (3 ) %Extr action Effi

ci ency 98. 3 Am en de d Effl ue nt % R ecove re d (% RSD) 65. 5 (3. 4) 8. 6 (7. 8) 74. 1 74. 9 (1. 5) %Extr action Effi

ci ency 88. 4 D5 Effl

ue nt % R ecove re d (% RSD) 71. 3 (4. 3) 14. 9 (1 .6) 86. 3 91. 8 (1. 5) %Extr action Effi

ci ency 82. 7 Am en de d Effl ue nt % R ecove re d (% RSD) 46. 5 (2. 8) 45. 5 (3 .7) 92. 0 91. 9 (1 1. 3) %Extr action Effi

ci ency 50. 6 D6 Effl

ue nt % R ecove re d (% RSD) 65. 7 (6. 7) 34. 6 (3) 100. 2 97. 9 (3. 6) %Extr action Effi

ci ency 65. 5 Am en de d Effl ue nt % R ecove re d (% RSD) 43. 2 (1. 9) 57. 1 (3 .4) 100. 3 92. 3 (1 5. 3) %Extr action Effi

ci ency 43. 1

% D5 Ex trac te d by LD PE 30 Mi nute s Vorte x 24. 6 60 Mi nute s Vorte x 46. 1 120 Mi nute s Vortex 60. 2 240 Mi nute s Vortex 85. 8HES Study Number 11936
- 108



### Page **26** of **32**

Table 5 . Linearity

Range of Solvent Standards Sol ve nt Conc en tra ti on, ng/g Equi

va le nt i n Sa mpl e μg/L % Re la ti

ve Error Co effi

ci en t of Determ ination D4 Std 1 0. 675 0. 0884 0. 0 1. 000 Std 2 1. 36 0. 178 0. 4 Std 3 4. 08 0. 535 0. 2 Std 4 9. 20 1. 21 0. 0 Std 5 18. 1 2. 37 3. 8 1. 000 Std 6 72. 9 9. 55 0. 4 Std 7 186 24. 4 1. 3 Std 8 501 65. 7 0. 1 Std 9 991 130 0. 0 D5 Std 1 0. 699 0. 0916 3. 9 1. 000 Std 2 1. 40 0. 184 0. 1 Std 3 4. 23 0. 554 1. 1 Std 4 9. 5 1. 25 0. 2 Std 5 18. 8 2. 46 0. 6 1. 000 Std 6 75. 5 9. 89 0. 9 Std 7 193 25. 2 0. 2 Std 8 519 68. 0 0. 1 Std 9 1026 134 0. 0 D6 Std 1 0. 604 0. 0791 3. 7 1. 000 Std 2 1. 21 0. 159 0. 2 Std 3 3. 65 0. 478 0. 9 Std 4 8. 23 1. 08 0. 1 Std 5 16. 2 2. 12 2. 8 1. 000 Std 6 65. 2 8. 55 2. 1 Std 7 166 21. 8 0. 2 Std 8 449 58. 8 0. 7 Std 9 887 116 0. 1HES Study Number 11936
- 108



### Page **27** of **32**

Table 6 . Recovery

of D4, D5 and D6 from Low and High Solids Matrix with LDPE Method LowSolids Hi gh Solids D4 D4 Spi ke d, μg/L Found, μg/L %R SD %Recove ry Spi ke d, μg /L Found, μg

/L %RS D %Recove ry 0. 000 0. 000 0 NA 0. 000 0. 085 6 NA 0. 493 0. 342 4 69 0. 493 0. 349 3 71 0. 987 0. 678 2 69 0. 987 0. 701 2 71 5. 74 3. 79 3 66 5. 74 3. 70 7 64 11. 5 7. 66 2 67 11. 5 7. 53 5 66 67. 3 45. 9 2 68 67. 3 46. 6 5 69 135 92. 6 2 69 135 96. 1 1 71 594 373 6 63 594 364 5 61 1189 497 8 42 1189 431 5 36

D5 D5 Spi ke d, μg/L Found, μg/L %R SD %Recove ry Spi ke d, μg /L Found, μg

/L %RS D %Recove ry 0. 000 0. 000 0 NA 0. 000 1. 504 15 NA 0. 512 0. 386 6 75 0. 512 0. 126 153 25 1. 02 0. 743 2 73 1. 02 0. 399 14 39 5. 95 4. 17 4 70 5. 95 3. 71 23 62 11. 9 8. 64 4 73 11. 9 8. 15 21 68 69. 7 52. 2 2 75 69. 7 46. 1 7 66 139 106. 5 2 76 139 96. 7 2 69 616 451 5 73 616 471 2 76 1232 870 3 71 1232 767 9 62

D6 D6 Spi ke d, μg/L Found, μg/L %R SD %Recove ry Spi ke d, μg /L Found, μg

/L %RS D %Recove ry 0. 000 0. 000 0 NA 0. 000 0. 256 14 NA 0. 454 0. 343 6 76 0. 454 0. 312 24 69 0. 908 0. 675 3 74 0. 908 0. 549 3 60 5. 28 3. 76 6 71 5. 28 3. 47 26 66 10. 6 8. 04 7 76 10. 6 7. 76 28 73 61. 9 49. 1 3 79 61. 9 43. 3 6 70 124 101 2 82 124 89. 0 2 72 547 437 5 80 547 477 2 87 1094 908 4 83 1094 816 18 75HES Study Number 11936
- 108



### Page **28** of **32**

Table 7 . Stability With and Without LDPE of Effluent Solutions Spiked with ~5 μg/L cVMS

Table 8 . Comparison of Headspace Storage versus LDPE with Effluents Spiked at ~5 μg/LcVMS

% Recove ry

(%RS D) % Recove ry (%RS D) D4- Am bi en t LDP E Control LDP E LHS Cont rol LHS D4- 5 °C LDP E Control LDP E LHS Control LHS Day 0 82 (4) 27 (1 6) 77 (1) 17 (12) Day 0 86 (1 ) 20 (1 7) 77 (4) 18 (1 3) Day 1 86 (3) 17 (8) 84 (2) 4 (21) Day 1 83 (11) 20 (7) 85 (1) 6 (5 ) Day 4 84 (4) 12 (3 2) 83 (3) 1 (6) Day 4 88 (2) 16 (5) 81 (2) 7 (4 1) Day 7 86 (2) 14 (3 6) 84 (2) 1 (21) Day 7 88 (2) 26 (8) 87 (1) 6 (1 1) Day 1 4 84 (2) 12 (6) 84 (2) 0 (1 03) Day 1 4 87 (8 ) 17 (9 7) 89 (2) 3 (1 ) D5- Am bie nt LDP E Control LDP E LHS Cont rol LHS D5 - 5 °C LDP E Control LDP E LHS Control LHS Day 0 78 (5) 66 (2) 81 (1) 58 (4) Day 0 84 (1 ) 61 (1 0) 76 (3) 59 (4) Day 1 88 (3) 61 (1) 87 (2) 40 (6) Day 1 81 (10) 60 (6) 83 (3) 42 (3) Day 4 90 (2) 56 (1 2) 89 (2) 27 (3) Day 4 88 (2) 60 (1) 80 (4) 44 (1 0) Day 7 93 (1) 60 (5) 89 (2) 20 (15) Day 7 84 (2) 70 (3) 83 (1) 41 (1 2) Day 1 4 94 (0) 48 (4) 92 (1) 5 (36) Day 1 4 87 (8 ) 65 (7) 89 (2) 37 (5) D6- Am bi en t LDP E Control LDP E LHS Cont rol LHS D6- 5 °C LDP E Control LDP E LHS Control LHS Day 0 71 (1 2) 84 (1) 80 (4) 79 (2) Day 0 83 (3 ) 85 (4) 74 (1 3) 78 (3) Day 1 67 (8) 78 (1) 71 (3) 71 (0) Day 1 74 (12) 79 (3) 72 (3) 69 (3) Day 4 77 (7) 72 (3) 77 (2) 60 (3) Day 4 72 (1 ) 77 (1) 65 (9) 68 (5) Day 7 80 (1) 71 (5) 78 (4) 47 (7) Day 7 70 (2) 82 (1) 70 (3) 64 (1 0) Day 1 4 83 (4) 51 (0) 88 (2) 17 (18) Day 1 4 76 (10) 78 (4) 77 (4) 62 (6)

% Recove ry

(% RS D) Day 0 Day

1 Day

4 D4 LDP E Am

bi en t 82 (4) 86 (3 ) 84 (4) LD PE5 C 86 (1 ) 83 (1 1) 88 (2 ) Headsp ace Vi

al 59 (3 ) 36 (1 3) D5 LDP E Am

bi en t 78 (5 ) 88 (3 ) 90 (4) LD PE5 C 84 (1 ) 81 (1 0) 88 (2 ) Headsp ace Vi

al 76 (3 ) 67 (1 3) D6 LDP E Am

bi en t 71 (1 2) 67 (8 ) 77 (7 ) LD PE5 C 83 (3 ) 74 (1 2) 72 (1 ) Headsp ace Vi

al 94 (5 ) 82 (9 )HES Study Number 11936
- 108



### Page **29** of **32**

Table 9 . Sample Stability Utilizing a Second Supplier of LDPE Film and LDPE Resin

Table 10 . Assessing the Limit of Detection, Method Detection Limit and Limit of Quantitation for D4, D5, and D6

* WWTP #2 D5 MDL and LOQ determined with n=6 % Recove ry

(% RSD ) % Recove ry (% RS D) D4- Am bi en t LDP E Resi

n D4- 5 ° C LD PE Resi n Day 0 87 (3 ) 86 (3 ) Da y 0 87 (3 ) 86 (3 ) Day 7 71 (3 ) 28 (1 ) Da y 7 77 (1 ) 47 (5 ) Day 1 4 58 (1 5) 25 (n =1) Day

1 4 81 (1 ) 49 (2) D5 - Am bi en t LDP E LD PE D5 - 5 ° C LD PE LD PE Day 0 91 (4) 92 (1 ) Da y 0 91 (4) 92 (1 ) Day 7 86 (7 ) 44 (1 ) Da y 7 90 (3 ) 65 (3 ) Day 1 4 83 (1 0) 32 (n =1) Day

1 4 90 (1 ) 61 (5 ) D6- Am bi en t LDP E LD PE D6- 5 ° C LD PE LD PE Day 0 97 (0) 98 (0) Da y 0 97 (0) 98 (0) Day 7 70 (1 6) 60 (1 ) Da y 7 91 (1 ) 91 (1 ) Day 1 4 86 (5 ) 43 (n =1) Day

1 4 91 (1 ) 81 (3 )

D4 , μg/L D5 , μg

/L D6 , μg/L WWTP # 1 LOD 0. 108 0. 037 0. 024 MDL 0. 019 0. 083 0. 044 LOQ 0. 047 0. 207 0. 111

WWTP # 2 LOD 0. 040 0. 094 0. 047 MDL 0. 032 0. 170* 0. 070 LOQ 0. 081 0. 424* 0. 175

Devel opme nt LOD 0. 006 0. 010 0. 019 MDL 0. 020 0. 157 0. 013 LOQ 0. 050 0. 393 0. 032

Se cond Suppl

ie r LDP E LOD 0. 011 0. 024 0. 008 MDL 0. 078 0. 128 0. 018 LOQ 0. 195 0. 321 0. 045

Average LOD 0. 041 0. 041 0. 025 MDL 0. 037 0. 123 0. 036 LOQ 0. 093 0. 307 0. 091HES Study Number 11936
- 108



### Page **30** of **32**

Table 1 1 . Results of LDPE Analy sis of WWTP #1 Aqueous Samples

Table 1 2 . Results of LDPE Analy sis of WWTP #2 Aqueous Samples

Table 1 3 . Results of Biosolid Samples WWTP # 1

Table 1 4 . WWTP #1 Results of Aqueous Samples Analyzed b y Direct Solvent Extraction Found, μg /L (% RSD ) D4 D5 D6 Infl ue nt 0. 178 (9 .4) 6. 738 (3 .2 ) 1. 225 (5 .8 ) Prim ar y Cl ari fi er 0. 113 (5 .1 ) 5. 421 (2 .4) 1. 015 (2 .8 ) Ae ra ti on Ba si n 0. 572 (5 .8 ) 25. 828 (7 .1 ) 5. 492 (9 .7 ) Se conda ry

Cl arif ie r <MD L <LO Q <MD L Effl ue nt <MD L 0. 292 (4 .8 ) <MD L MDL 0.019 0.083 0.044 LOQ 0.047 0.207 0.111
Found, μg /L (% RSD ) D4 D5 D6 Infl ue nt 0. 176 (4 .2 ) 8. 150 (3 .0 ) 1. 928 (6 .4) Effl ue nt <MD L 0. 179 (2 8. 1) <MD L MDL 0.032 0.170 0.070 LOQ 0.081 0.424 0.175
Found, μg /k g (% RSD ) D4 D5 D6 Po st Di ge ste r 17. 8 (2 .4) 933. 9 (0 .2 ) 213. 6 (3 .9 ) Sl udge from Pr imar

y Cl arif ie r 35. 0 (2 .6 ) 676. 9 (1 .9 ) 141. 0 (2 .9 ) Sl udge from DAF 26. 1 (1 .1 ) 1,369. 1 (0 .2 ) 317. 4 (4 .4) Sl udge from Fi

na l Cl ari fi er 4. 8 (1 .5 ) 210. 8 (0 .4) 52. 2 (4 .7 ) Ski mme d from Primar

y Cl arif ie r 2. 3 (1 3. 9) 27. 3 (1 5. 6) 7. 7 (1 6. 5) DAF= Di sso lved Ai r F lota ti on
Found, μg /L (% RS D) D4 D5 D6 Infl ue nt 0. 345 (1 .4) 7. 145 (0 .5 ) 1. 274 (0 .7 ) Primary Cl ari fi er 0. 275 (1 .8 ) 5. 402 (0 .8 ) 1. 056 (3 .3 ) Aer ation Ba si

n 0. 663 (0 .6 ) 22. 794 (0 .8 ) 5. 353 (0 .2 ) Se condary

Cl ari fi er 0. 096 (4 .8 ) 0. 259 (6 .6 ) 0. 087 (6 .8 ) Ef fl ue nt 0. 096 (1 .1 ) 0. 353 (4 .8 ) 0. 111 (9 .5 )HES Study Number 11936
- 108



### Page **31** of **32**

Table 1 5 . WWTP #1
- Percent Difference Comparing LDPE Method to Direct Solvent

Extraction of Aqueous Samples

Table 1 6 . WWTP #2 Results of Aqueous Samples Analyzed by Direct Solvent Extraction

Table 1 7 . WWTP #2
- Percent Difference Comparing LDPE Method to Direct Solvent

Extraction of Aqueous Samples % Di fference ( Ab so lu

te ) D4 D5 D6 Infl ue nt 93. 8 6. 0 4. 0 Primary Cl ari fi er 143. 4 0. 4 4. 0 Aer ation Ba si

n 15. 9 11. 7 2. 5 Se condary

Cl ari fi er NA 45. 5 NA Ef fl ue nt NA 20. 9 NA NA= Not Appl ic ab le d ue to LD PEresult s b el

owMDL

Found, μg /L (% RS D) D4 D5 D6 Infl ue nt 0. 216 (0 .9 ) 6. 370 (1 .0 ) 1. 576 (1 .1 ) Ef fl ue nt 0. 090 (2 .5 ) 0. 316 (1 .3 ) 0. 173 (8 .7 )

% Di fference ( Ab so lu

te ) D4 D5 D6 Infl ue nt 22. 7 21. 8 18. 3 Ef fl ue nt NA 76. 5 NA NA= Not Appl ic able d ue to LD PEresult s b el

owMDLHES Study Number 11936
- 108



### Page **32** of **32**

**FI GURES**  Figure 1
. Stabil i ty

of cVMS in Samples with LDPE, without LDPE, and Low Headspace

0 10 20 30 40 50 60 70 80 90 100

0 2 4 6 8 10 12 14**% R** **e c** **o v** **e r** **y**

**Days**

D4 - Am bie nt Stability

Cont rol LDPE Cont rol LH S LDPEL HS 0 10 20 30 40 50 60 70 80 90 100

0 2 4 6 8 10 12 14**% R** **e c** **o v** **e r** **y**

**Days**

D4 - 5 ° C Stability

Cont rol LD PE Cont rol LHS LD PE LHS

0 10 20 30 40 50 60 70 80 90 100

0 2 4 6 8 10 12 14**% R** **e c** **o v** **e r** **y**

**Days**

D5 - Ambie nt Stability

Control LD PE Control LH S LD PE LHS 0 10 20 30 40 50 60 70 80 90 100

0 2 4 6 8 10 12 14**% R** **e c** **o v** **e r** **y**

**Days**

D5 5 ° C Stability

Control LD PE Control LHS LD PE LHS

0 10 20 30 40 50 60 70 80 90 100

0 2 4 6 8 10 12 14**% R** **e c** **o v** **e r** **y**

**Days**

D6 - Ambient Stability

Cont rol LD PE Cont rol LHS LD PEL HS 0 10 20 30 40 50 60 70 80 90 100

0 2 4 6 8 10 12 14**% R** **e c** **o v** **e r** **y**

**Days**

D6 5 ° C Stability

Cont rol LDPE Cont rol LH S LDPEL HS