HES Study Number 11936-108

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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 2013
HES Study Number 11936-108

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TABLE OF CONTENTS

TABLE OF CONTENTS ................................................................................................................ 2

ABSTRACT.................................................................................................................................... 4

SIGNATURES AND APPROVAL ................................................................................................ 5

OBJECTIVE.................................................................................................................................... 6

TEST ARTICLE SPECIFICATIONS............................................................................................. 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 DISCUSSION.................................................................................................... 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................................................................................................................................... 28
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Table 9. Sample Stability Utilizing a Second Supplier of LDPE Film and LDPE 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

FIGURES ...................................................................................................................................... 32

Figure 1. Stability of cVMS in Samples with LDPE, without LDPE, and Low Headspace32
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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 

waterways 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 octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5), and 

dodecamethylcyclohexasiloxane (D6) in wastewater treatment plant influent, effluent and 

surface waters. This method utilizes low density polyethylene (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 

analyzed by gas chromatography mass spectrometry. The benefits of the method are its 

ease of sample collection and analysis 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 days at room temperature conditions. 

Linearity 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.
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SIGNATURES AND APPROVAL

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

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OBJECTIVE

The objective of this study is to develop an analytical method for determining the 

collection, storage and analysis procedures for the quantification of 

octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5), and 

dodecamethylcyclohexasiloxane (D6) in waste water treatment plant (WWTP) influent, 

effluent and surface water samples.

TEST ARTICLE SPECIFICATIONS

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 1a

 Identification: Octamethylcyclotetrasiloxane (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

 Identification: Octamethylcyclotetrasiloxane (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 2a

 Identification: 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 3a

 Identification: Dodecamethylcyclohexasiloxane (D6)

 CAS Number: 540-97-6

 Lot Number: 0005578887 (supplied as Dow CorningTM 246 Fluid)

 Expiration Date: 05Oct2011

 Purity: 97.3 % by GC-FID
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e. Reference Article 3b

 Identification: 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

EXPERIMENTAL

A. Background

Cyclic volatile methyl siloxanes (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 subsequently 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 able to accurately quantify 

cVMS in aqueous matrices. 

The primary objective of the current study was to develop a method to preserve 

and accurately 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), 

decamethylcyclopentasiloxane (D5), and dodecamethylcyclohexasiloxane (D6) -

are volatile, have low solubility in water, and very high Henry’s law constants, 

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 6.121, respectively - indicating a strong tendency to partition onto 

organic matter. Methods for collection and analysis of aqueous matrices must take 

into account the dissolved and labile fractions of cVMS that may readily 

evaporate, as well as those fractions that are associated with the organic content 

within the water column. Consequently, methods for collection and analysis 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 analyte to the 
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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 

polytetrafluoroethylene (PTFE) lined lid. The jars were prepared by wrapping 

PTFE tape around the threads of the jar to further inhibit losses 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 

stainless 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 (effectively collecting 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 with 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 packs 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 mL of 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 

Fisher 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 analysis by gas chromatography/massspectrometry (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-mL scintillation vial 

followed by 2.5 mL of acetonitrile and 5 mL of ISWS. The mixture was vortexed 

on high for 30 minutes and centrifuged at 2000 rpm (1308 relative centrifugal 

force) for five minutes. The extract was removed and added to another vial. An 

additional 5 mL of hexanes was added to the sample containing acetonitrile and 
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vortexed and centrifuged as described above. The extract was removed and 

combined with the original extract and an aliquot was analyzed by GC-MS. 

Samples were analyzed with triplicate preparations.

Direct Solvent Extraction Sample Preparation

From the large aliquot of sample that was retained during collection, 50 mL 

aliquots of sample were weighed into 4-oz jars along with 10 mL of the ISWS. 

The mixtures were vortexed on high for 30 minutes and the phases allowed to 

separate. An aliquot of the organic layer was removed for analysis by GC-MS. 

Samples were analyzed with triplicate preparations.

LDPE Sample Analysis

Samples were separated and quantified on a GCMS utilizing a DB-Wax etr or 

DB-5ms chromatography 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 ISWS and reanalyzed. During analysis, the samples were bracketed by

solvent blanks and standards to ensure the absence of carryover and that the 

standard curve remained within control limits. 

In order to ensure a valid calibration curve and analysis, the coefficient of 

determination, R2, of greater than 0.995 was the criterion 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.
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Chromatographic Conditions

Equipment Agilent 7890 GC coupled with 5975 

MSD and a CTC Analytics 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 analysis 

from the point of sample collection to analysis. Potential problems faced when 

analyzing for trace levels of cVMS in water samples are contamination from the 

environment, analyst, 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 well as analyte 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 capped 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 remained 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 opened during the time of sampling to 

determine if there was ambient contamination that could possibly deposit into the 

samples at that time. These blanks were closed after sampling and analyzed with 

the collected samples.
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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 μL of 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/L for site #1 and 110 μg/L for site #2). This spike’s purpose was to show that 

the analyte was sufficiently retained by the sample container/LDPE and no 

degradation of the analytes occurred due to the matrix or the collection

conditions. 

D. High Organic Content Samples

Relatively high log Koc and Kaw5values are found for cVMS, indicating that 

they readily partition to suspended matter/organic content in the water column as 

well as evaporate from the aqueous sample. The effluent 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/L cVMS. 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 mL of 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 time 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 tetrahydrofuran from ~0.5 to 1100 μg/L making sure to 

place the spikes directly 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 relatively low amount as 10 – 30 mg/L is 

typical6. Since the suspended solids are low that means the organic content is also 
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relatively 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 system 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 of the 4 oz jar jar, 

while the sample consumes nearly 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 processed 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-mL scintillation vial for extraction into the hexanes 

based internal standard solution. The vials were then vortexed for 30 minutes. 

Samples that were not stored with LDPE were processed by liquid-liquid 

extraction into 10 mL of the hexanes based internal standard solution and 

vortexed 30 minutes.

Another form of collection noted in literature is the use of 100-mL headspace 

vials with sample work up using a membrane assisted solvent extraction system7. 

As part of this stability study, effluent was added to the 100-mL headspace 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 and Film, 2 mil LDPE ClearBase 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 each sample. 

Each pellet was ellipsoid in shape with an approximate surface area of 49.3 mm2.
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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 analysis to assess the 

suitability of the method and equipment at the time of analysis 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 greater 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 solutions8,9. 

The method detection limit (MDL) was defined as the level of analyte in a sample 

matrix, that when processed through the complete analytical method, can be 

measured and reported with 99% probability that it is greater than zero and was 

calculated as tn-1, α1=0.01 x s where s is standard deviation of seven replicate 

preparations and analysis of effluent. 

The limit of quantitation of a method was defined as the minimum level of analyte 

in a sample matrix that can be measured and accurately 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 analysis 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 passes 

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 prior 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 analyzed.
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DATA 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 analytical 

effects. The values from samples collected from the WWTP sites were corrected

using the values from internal standard blanks. Validation and developmental 

samples were corrected for matrix blanks, either effluent or milli-Q waterwhichever 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 counterparts that were used as internal 

standards. 

Exponential regression equation of the line and equation for sample analysis

 =  +  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, ng
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Percent Recovery (%Rec)

% =





 100 

Where:

%Rec = Percent recovery, %

F = Found test article, ng

T = Theoretical test article, ng

RESULTS AND DISCUSSION

A. Quality Control Program

The QC program as described above was executed at both WWTP collection 

sites. All QC samples were prepared and analyzed 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 analyzed for WWTP #2 

were all below the defined LOD as shown in Table 2 for the respective analytes 

except one value that was between the MDL and the LOD. The largest LOD for 

all analytes was 0.094 μg/L. The MDL for one analyte 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 blanks all had quantifiable amounts 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 found 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 only open during 

the sample collection and not during the spiking of the field spikes.
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B. High Organic Content Samples

Two different matrix sets were analyzed with differing amounts of 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/L cVMS. All samples were prepared and analyzed in triplicate. The samples 

were analyzed such 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 analyzed where the LDPE and aqueous phase were 

extracted together.

The data shown in Table 3 clearly 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 75%, 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 lost 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 by the percent extraction efficiency noted in Table 3. The lower 

extraction 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, respectively. 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 tightly 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 D61. Also noteworthy is that the 

total recovery for the samples where the LDPE and remaining aqueous phase

were extracted separately into hexanes is very similar to the samples where the 

LDPE and aqueous phase were extracted together into 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 240 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 extracted by the LDPE increased over time starting 

at 24.6% at 30 minutes to 85.8% after 240 minutes of vortexing. This 
HES Study Number 11936-108

Page 17 of 32

demonstrates that the D5 does preferentially partition from the organic carbon to 

the LDPE, but the process requires time and energy. It also shows the potential 

for LDPE to be used as a means to measure the freely labile portion of D5 in these 

samples, but would require further study.

C. Linearity

Linearity of this methodology was established by using the prepared solvent 

standards. 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 established 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 calculate 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 found 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/L for 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 recovery of this method was also demonstrated on an effluent matrix which 

had been amended with post-digested biosolids to a solids content of ~47 mg/L. 

As a result 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, essentially limiting the lower range of the recovery study to this level. The 

other cVMS, D4 and D6 were not similarly 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 system to sequester ~5 μg/L cVMS was 

demonstrated for 14 days at both ambient temperatures and at 5 °C. It should be 

noted that since these samples were processed differently and the LDPE was

separately extracted into the internal standard solution, these results show that the 

LDPE is the keeper of the analytes in this low suspended solids effluent matrix. 

The stability results can be seen graphically 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 headspace (LHS), 

and without LDPE LHS. In all 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 recoveries were all ≤ 

27%. An advantage was also noted for D5 where the minimum recovery over 14 

days was 76%. For D6 at 5 °C, all samples were above 62% recovery at each 

time point for 14 days. 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 hypothesized 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 fraction that may have sorbed to the lid would not have been 

accounted for. This hypothesis was not evaluated in the laboratory.

The data for the 100-mL headspace vial experiment is shown in Table 8. The 

headspace storage method had considerable loss of D4 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 

acceptable at 76% after 1 day of storage and 67% after 4 days. The headspace 

storage method seemed to perform better than the LDPE method with recoveries 

of 94% and 82% for day 1 and day 4, respectively, while the LDPE method 

dipped to 67% recovery. Due to the 40% loss in 24 hours of 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 clearly shows that the LDPE film helps prevent 

significant volatilization in samples that are spiked with cVMS, future 

experiments may want to evaluate systems that are in equilibrium as these may

have a longer shelf life than do samples whose equilibrium are perturbed by 
HES Study Number 11936-108

Page 19 of 32

spiking additional cVMS. This perturbation may cause loss of the unbound 

cVMS while the sample 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 conditions. The time points prepared were 0 day, 7 days, and 14 

days. 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 days of 

storage. At ambient temperature the film did not perform as well for D4 through 

14 days as shown by the 58% recovery, but D5 and D6 results were acceptable at 

83 and 86% recovery, respectively. However, after 7 days the D4 results at 

ambient temperature was acceptable with 71% recovery. The second source of 

the film performed well for all three analytes after 7 days of storage at ambient 

temperature. Under refrigerated temperature, 5 °C, the film showed good 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 adequately help the stability of the sample as the recoveries were 28 

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

43%. At 5 °C, the samples fared slightly better but not sufficient to use as a 

means for storage. After 7 days 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 times during this study and the results are tabulated in Table 10. 

The LOD was analyzed using 7 replicates of LDPE extractions into the ISWS. 

The MDL and LOQ were analyzed from 7 replicate samples of effluent with the 

exception of the MDL and LOQ for D5 from WWTP #2 which used 6 replicates 

due to an outlier result10. Insufficient effluent was collected from WWTP #1 so

the MDL and 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 stock as used in the samples to ensure an accurate comparison. In one 

case the LOD for D4 was significantly greater than the MDL for undetermined 
HES 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 Table 10 and used in calculating the average LOD.

Using the averages, the limits of detection for D4, D5, and D6 were 0.041, 0.041, 

and 0.025 μg/L, respectively. Similarly, the method detection limits were 0.037, 

0.123, and 0.036 μg/L with a limit of quantitation of 0.093, 0.307, and 0.091

μg/L, respectively. 

H. LDPE Mode of Action

The mode of action for the storage of cVMS by 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 sample and or the sample surface. The 

surface area of the LDPE film is 10323 mm2accounting for the two primary 

surfaces of all eight squares, with each square having a surface area of 645 mm2

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 mm2for 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 mL and in the 4 oz jars utilized, the surface 

area of the exposed sample is 128 mm2. 

As shown above the surface area of the LDPE film is far greater than the surface 

area of the sample; however the film rarely 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 nearly 200 pellets to get 

nearly the same surface area, however this may present other sample preparation 

and extraction problems because this would also mean the amount of pellets 

would be 6.8g, potentially interfering with extraction mixing.

It is not uncommon for WWTP to collect samples using plastic containers and 

tubing. When analyzing 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 analyzed 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 collected from the influent, primary settling tank, aeration basin, 

secondary clarifier, and effluent for analysis 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 of the WWTP process as the hydraulic 

retention time of each portion of the process was not considered. The results from 

the analysis by the LDPE method can be found in Table 11. 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 analytes 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, 

respectively, 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 analysis. All 

samples were collected in an afternoon and as in WWTP #1, no conclusions can 

be drawn about removal efficiency. The results from analysis by the LDPE 

method are shown in Table 12. The results in the table are corrected for LDPE 

blanks. 

The influent samples were found to contain 0.176 μg/L, 8.150 μg/L, and 1.928 

μg/L of D4, D5 and D6, respectively, and the effluent 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 analyzed in triplicate using the biosolids method 

described above. As noted in Table 13, a significant portion of cyclics (relative to 

the water matrices) 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

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Direct Solvent Extraction of Aqueous Samples – Municipality #1

The results from the direct solvent extraction of the aqueous samples are 

displayed in Table 14. This methodology was not validated and a method 

detection limit was not estimated, but rather used as a comparative analysis. The 

comparison of the direct solvent extraction to the LDPE method is shown in Table 

15. 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 by 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 method. 

Direct Solvent Extraction – Municipality #2

The direct solvent extraction results and comparison of the samples from 

WWTP#2 are shown in Tables 16 and 17, respectively. Similar to the results 

from WWTP#1, all results above 0.5 μg/L were below 22 % relative difference. 

CONCLUSIONS

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 the sample was demonstrated for 14 days 

at 5 °C using both sources of LDPE film. The stability was demonstrated for 7 

days at ambient laboratory conditions. LDPE resin pellets did not sufficiently

retain the spiked cVMS.

The analytical 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 limits for D4, 

D5, and D6 were 0.037, 0.123, and 0.036 μg/L, respectively.

Samples containing a higher amount of solids affected 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 of 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 Treatment 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. Method 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 cyclic 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 Health Assn: 2005.

10. Barnett, V.; Lewis, T. Outliers in Statistical Data (3rd edition), Wiley 1994.
HES Study Number 11936-108

Page 24 of 32

TABLES

Table 1. Quality Control Blank Samples 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 

(%RSD)

D6, μg/L 

(%RSD)

WWTP #1 Trip Blank <MDL <LOQ <MDL

Influent Blank 0.328 (97.0) 0.223 (45.8) <LOQ

Effluent Blank 1.076 (140.8) 0.397 (104.8) <LOQ

MDL 0.019 0.083 0.044

LOQ 0.047 0.207 0.111

WWTP #2 Trip Blank <LOD <LOD <LOD

Influent Blank <LOD <LOD <LOD

Effluent Blank <LOD <MDL <LOD

LOD 0.040 0.094 0.047

MDL 0.032 0.170* 0.070

D4, % Recovery

(%RSD)

D5, Recovery

(%RSD)

D6, Recovery

(%RSD)

WWTP #1 Influent Field Spike 83.3 (4.0) 90.0 (3.1) 91.9 (1.0)

Effluent Field Spike 82.7 (0.5) 81.6 (0.9) 81.8 (1.4)

High Spike Check 85.7 (3.1) 90.6 (1.8) 91.7 (0.9)

Low spike Check 86.8 (2.4) 90.1 (0.8) 86.2 (4.9)

High/Influent Spike ~3800 μg/L

Low/Effluent Spike ~3.6 μg/L

WWTP #2 Influent Field Spike 87.6 (1.0) 97.0 (1.3) 92.7 (1.1)

Effluent Field Spike 87.3 (1.6) 94.6 (0.9) 90.5 (0.6)

High Spike Check 98.8 (0.3) 97.5 (0.3) 97.7 (0.1)

Low spike Check 98.9 (0.8) 107.2 (13.3) 99.2 (1.3)

High/Influent Spike ~110 μg/L

Low/Effluent Spike ~4.3 μg/L
HES Study Number 11936-108

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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

LDPE 

Extraction

Water After LDPE 

Extraction Total Recovered

Combined 

Extraction

D4 Effluent

% Recovered (%RSD) 47.5 (3.2) 0.8 (16) 48.3 47.7 (3)

%Extraction Efficiency 98.3

Amended Effluent

% Recovered (%RSD) 65.5 (3.4) 8.6 (7.8) 74.1 74.9 (1.5)

%Extraction Efficiency 88.4

D5 Effluent

% Recovered (%RSD) 71.3 (4.3) 14.9 (1.6) 86.3 91.8 (1.5)

%Extraction Efficiency 82.7

Amended Effluent

% Recovered (%RSD) 46.5 (2.8) 45.5 (3.7) 92.0 91.9 (11.3)

%Extraction Efficiency 50.6

D6 Effluent

% Recovered (%RSD) 65.7 (6.7) 34.6 (3) 100.2 97.9 (3.6)

%Extraction Efficiency 65.5

Amended Effluent

% Recovered (%RSD) 43.2 (1.9) 57.1 (3.4) 100.3 92.3 (15.3)

%Extraction Efficiency 43.1

% D5 Extracted 

by LDPE

30 Minutes Vortex 24.6

60 Minutes Vortex 46.1

120 Minutes Vortex 60.2

240 Minutes Vortex 85.8
HES Study Number 11936-108

Page 26 of 32

Table 5. Linearity Range of Solvent Standards

Solvent 

Concentration, ng/g

Equivalent in 

Sample μg/L

% Relative

Error

Coefficient of

Determination

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 0.1
HES 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

Low Solids High Solids

D4 D4

Spiked, μg/L Found, μg/L %RSD %Recovery Spiked, μg/L Found, μg/L %RSD %Recovery

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

Spiked, μg/L Found, μg/L %RSD %Recovery Spiked, μg/L Found, μg/L %RSD %Recovery

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

Spiked, μg/L Found, μg/L %RSD %Recovery Spiked, μg/L Found, μg/L %RSD %Recovery

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 75
HES Study Number 11936-108

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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/L cVMS

% Recovery (%RSD) % Recovery (%RSD)

D4 - Ambient LDPE Control LDPE LHS Control LHS D4 - 5 °C LDPE Control LDPE LHS Control LHS

Day 0 82 (4) 27 (16) 77 (1) 17 (12) Day 0 86 (1) 20 (17) 77 (4) 18 (13)

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 (32) 83 (3) 1 (6) Day 4 88 (2) 16 (5) 81 (2) 7 (41)

Day 7 86 (2) 14 (36) 84 (2) 1 (21) Day 7 88 (2) 26 (8) 87 (1) 6 (11)

Day 14 84 (2) 12 (6) 84 (2) 0 (103) Day 14 87 (8) 17 (97) 89 (2) 3 (1)

D5- Ambient LDPE Control LDPE LHS Control LHS D5 - 5 °C LDPE Control LDPE LHS Control LHS

Day 0 78 (5) 66 (2) 81 (1) 58 (4) Day 0 84 (1) 61 (10) 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 (12) 89 (2) 27 (3) Day 4 88 (2) 60 (1) 80 (4) 44 (10)

Day 7 93 (1) 60 (5) 89 (2) 20 (15) Day 7 84 (2) 70 (3) 83 (1) 41 (12)

Day 14 94 (0) 48 (4) 92 (1) 5 (36) Day 14 87 (8) 65 (7) 89 (2) 37 (5)

D6 - Ambient LDPE Control LDPE LHS Control LHS D6 - 5 °C LDPE Control LDPE LHS Control LHS

Day 0 71 (12) 84 (1) 80 (4) 79 (2) Day 0 83 (3) 85 (4) 74 (13) 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 (10)

Day 14 83 (4) 51 (0) 88 (2) 17 (18) Day 14 76 (10) 78 (4) 77 (4) 62 (6)

% Recovery (%RSD)

Day 0 Day 1 Day 4

D4 LDPE Ambient 82 (4) 86 (3) 84 (4)

LDPE 5 C 86 (1) 83 (11) 88 (2)

Headspace Vial 59 (3) 36 (13)

D5 LDPE Ambient 78 (5) 88 (3) 90 (4)

LDPE 5 C 84 (1) 81 (10) 88 (2)

Headspace Vial 76 (3) 67 (13)

D6 LDPE Ambient 71 (12) 67 (8) 77 (7)

LDPE 5 C 83 (3) 74 (12) 72 (1)

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

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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

% Recovery (%RSD) % Recovery (%RSD)

D4 - Ambient LDPE Resin D4 - 5 °C LDPE Resin

Day 0 87 (3) 86 (3) Day 0 87 (3) 86 (3)

Day 7 71 (3) 28 (1) Day 7 77 (1) 47 (5)

Day 14 58 (15) 25 (n=1) Day 14 81 (1) 49 (2)

D5- Ambient LDPE LDPE D5 - 5 °C LDPE LDPE

Day 0 91 (4) 92 (1) Day 0 91 (4) 92 (1)

Day 7 86 (7) 44 (1) Day 7 90 (3) 65 (3)

Day 14 83 (10) 32 (n=1) Day 14 90 (1) 61 (5)

D6 - Ambient LDPE LDPE D6 - 5 °C LDPE LDPE

Day 0 97 (0) 98 (0) Day 0 97 (0) 98 (0)

Day 7 70 (16) 60 (1) Day 7 91 (1) 91 (1)

Day 14 86 (5) 43 (n=1) Day 14 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

Development

LOD 0.006 0.010 0.019

MDL 0.020 0.157 0.013

LOQ 0.050 0.393 0.032

Second Supplier LDPE

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 0.091
HES Study Number 11936-108

Page 30 of 32

Table 11. Results of LDPE Analysis of WWTP #1 Aqueous Samples

Table 12. Results of LDPE Analysis of WWTP #2 Aqueous Samples

Table 13. Results of Biosolid Samples WWTP #1

Table 14. WWTP #1 Results of Aqueous Samples Analyzed by Direct Solvent 

Extraction

Found, μg/L (%RSD)

D4 D5 D6

Influent 0.178 (9.4) 6.738 (3.2) 1.225 (5.8)

Primary Clarifier 0.113 (5.1) 5.421 (2.4) 1.015 (2.8)

Aeration Basin 0.572 (5.8) 25.828 (7.1) 5.492 (9.7)

Secondary Clarifier <MDL <LOQ <MDL

Effluent <MDL 0.292 (4.8) <MDL

MDL 0.019 0.083 0.044

LOQ 0.047 0.207 0.111

Found, μg/L (%RSD)

D4 D5 D6

Influent 0.176 (4.2) 8.150 (3.0) 1.928 (6.4)

Effluent <MDL 0.179 (28.1) <MDL

MDL 0.032 0.170 0.070

LOQ 0.081 0.424 0.175

Found, μg/kg (%RSD)

D4 D5 D6

Post Digester 17.8 (2.4) 933.9 (0.2) 213.6 (3.9)

Sludge from Primary Clarifier 35.0 (2.6) 676.9 (1.9) 141.0 (2.9)

Sludge from DAF 26.1 (1.1) 1,369.1 (0.2) 317.4 (4.4)

Sludge from Final Clarifier 4.8 (1.5) 210.8 (0.4) 52.2 (4.7)

Skimmed from Primary Clarifier 2.3 (13.9) 27.3 (15.6) 7.7 (16.5)

DAF = Dissolved Air Flotation

Found, μg/L (%RSD)

D4 D5 D6

Influent 0.345 (1.4) 7.145 (0.5) 1.274 (0.7)

Primary Clarifier 0.275 (1.8) 5.402 (0.8) 1.056 (3.3)

Aeration Basin 0.663 (0.6) 22.794 (0.8) 5.353 (0.2)

Secondary Clarifier 0.096 (4.8) 0.259 (6.6) 0.087 (6.8)

Effluent 0.096 (1.1) 0.353 (4.8) 0.111 (9.5)
HES Study Number 11936-108

Page 31 of 32

Table 15. WWTP #1 - Percent Difference Comparing LDPE Method to Direct Solvent 

Extraction of Aqueous Samples

Table 16. WWTP #2 Results of Aqueous Samples Analyzed by Direct Solvent 

Extraction

Table 17. WWTP #2 - Percent Difference Comparing LDPE Method to Direct Solvent 

Extraction of Aqueous Samples

% Difference (Absolute)

D4 D5 D6

Influent 93.8 6.0 4.0

Primary Clarifier 143.4 0.4 4.0

Aeration Basin 15.9 11.7 2.5

Secondary Clarifier NA 45.5 NA

Effluent NA 20.9 NA

NA= Not Applicable due to LDPE results below MDL

Found, μg/L (%RSD)

D4 D5 D6

Influent 0.216 (0.9) 6.370 (1.0) 1.576 (1.1)

Effluent 0.090 (2.5) 0.316 (1.3) 0.173 (8.7)

% Difference (Absolute)

D4 D5 D6

Influent 22.7 21.8 18.3

Effluent NA 76.5 NA

NA= Not Applicable due to LDPE results below MDL
HES Study Number 11936-108

Page 32 of 32

FIGURES

Figure 1. Stability of cVMS in Samples with LDPE, without LDPE, and Low 

Headspace

0

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0 2 4 6 8 10 12 14

% Recovery

Days

D4 - Ambient Stability

Control

LDPE

Control LHS

LDPE LHS

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% Recovery

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D4 - 5° C Stability

Control

LDPE

Control LHS

LDPE LHS

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% Recovery

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D5 - Ambient Stability

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LDPE

Control LHS

LDPE LHS

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0 2 4 6 8 10 12 14

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LDPE LHS

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D6 - Ambient Stability

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LDPE LHS

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% Recovery

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D6 5° C Stability

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LDPE LHS