Testing Standard for the Collection, Sample Handling, Transfer and Analysis of Biota Samples for D4

August 16, 2013

Silicones Environmental, Health and Safety Center 1

Synopsis: 

This testing standard provides guidelines for the collection and analysis of environmental samples for D4

in support of a Testing Program detailed in this Enforceable Consent Agreement (ECA). The purpose of 

the Testing Program is to “...develop environmental testing data for D4 in the vicinity of manufacturing 

and processing (including product formulating) sites and POTWs in the U.S. so that exposures and risks 

to sediment and aquatic organisms can be assessed.” This standard cites the requirements for the 

sampling of biota as described in Section VII of the ECA and Appendices 1, 2 and 3, and identifies specific 

guidelines and references that will be used in the preparation of site-specific Sampling Plans for the 

collection and analysis of biota samples, which will be included in a Study Plan for the Testing Program. 

Requirements for sampling biota: 

Per Appendix 1, Environmental Testing at D4 Direct Discharge Sites, Appendix 2, Environmental Testing 

for Indirect Discharge Sites, and Appendix 3, POTWs Not Receiving D4 from Industrial Facilities:

“ Biota sampled shall include benthic species collected in sediment of the receiving water body and, 

if reasonably practicable, two species of fish...in the receiving water body.”

Establishment of the location for benthic organism collection: 

At each site (i.e., POTW and direct discharge sites) benthic organisms should be collected from sediment 

located as close as possible to the wastewater outfall release point. However, because sediments are 

expected to be found only in deposition zones within the receiving stream, the specific sampling 

location must also be within a deposition zone and be of sufficient area to allow collection of both 

sediment and benthos samples. Accordingly, samples of benthic organisms should be collected from the 

deposition zone nearest the facility wastewater outfall release point. This location should be the same 

as the location where sediments are collected for D4 analysis. If insufficient numbers or mass of benthic 

organisms can be collected from the selected deposition zone, sampling will continue at subsequent

downstream deposition zone(s) until sufficient numbers or mass are obtained. 

Determination of the location of the nearest deposition zone of sufficient size to take samples of benthic 

organisms and sediment is a facility-specific inquiry. The facility operator or permitting authority should 

be consulted, as appropriate, to determine if the deposition zone has been established. For cases in 

which the nearest deposition zone is not known, it should be determined in the field by reconnaissance 

prior to the actual collection of samples. For each site, establishment of the deposition zone by field 

reconnaissance shall be detailed in the site-specific Sampling Plans.
Testing Standard for the Collection, Sample Handling, Transfer and Analysis of Biota Samples for D4

August 16, 2013

Silicones Environmental, Health and Safety Center 2

Establishment of the location for collection of fish: 

At each site (i.e., POTW or direct discharge facility), fish should be collected as close as possible to the 

discharge point provided adequate mixing of the discharge and receiving water has occurred. To satisfy 

these two requirements, fish shall be collected at the end of the mixing zone in locations where fish may 

be expected to be captured (e.g., near the shoreline). 

Determination of a mixing zone is a facility-specific inquiry. For each facility, the mixing zone may have 

been established with the applicable wastewater permitting authority. The facility operator and 

permitting authority should be consulted, as appropriate, to determine if a mixing zone has been 

established. For cases in which the mixing zone is not known to the facility operator, it should be 

determined in the field using appropriate measurement or modeling techniques prior to the actual 

collection of samples. Methods for the analysis of mixing zones are presented in USEPA (1991, Section 

4.4, Mixing Zone Analyses) and USEPA (2006, Modeling Documents section, pp.9-13) (see Annexes 1 and 

2) and should be utilized, as appropriate. Each site-specific Sampling Plan shall include a discussion of 

the mixing zone in the receiving water, including the basis for the establishment of the specified zone. 

Special considerations for the sampling and handling of biota samples for D4: 

Cyclic volatile methyl siloxane (cVMS) materials, such as D4, are widely used in consumer products and 

may be present in personal care products, commercial products, and lubricants, and may be 

components of antifoams widely used in waste water treatment plants. To avoid the potential for 

sample contamination during collection, processing, storage, and analysis for D4 in environmental 

matrices, appropriate quality control measures should be implemented to the extent possible in the 

field. For example, field crew shall refrain from using any personal care products (e.g. , sun-block, sunscreen, hand lotion, antiperspirants, etc.) while preparing for or conducting any field activities to 

minimize the possibility for contamination with cVMS materials. Appropriate requirements for collecting 

and handling biota samples that comply with the special considerations related to D4 shall be specified 

in the site-specific Sampling Plan. 

Collection of benthic organism samples for analysis of D4: 

On the day of field sampling at a given site, two (n=2) composited samples of benthic organisms shall be 

collected in accordance with the sample collection procedures set out in the site-specific Sampling Plan. 

Sediments containing benthic organisms will be collected using one or more methods specified in the 

site-specific sampling plan such that sufficient numbers or mass of organisms are collected for the 

analysis of D4. Benthic organisms will be separated from the sediment and composite samples of the 

organisms shall be prepared. The sample collection procedures will be based, as appropriate, on 

techniques described in USEPA (2003a, Sections 5.2 Sampling for Community Samples and 5.3 Sampling 

for Analytical Measurements) and Powell and Woodburn (2009, Sample Collection, p. 9, Analysis of 
Testing Standard for the Collection, Sample Handling, Transfer and Analysis of Biota Samples for D4

August 16, 2013

Silicones Environmental, Health and Safety Center 3

Cyclic Volatile Methylsiloxane (cVMS) Materials section, Extraction of Fish and Benthic 

Macroinvertebrates sub-section, p. 13, and Quality Control, p. 14) (see Annexes 3-4). Sample collection 

procedures as well as specific requirements for sample container preparation, sample preservation, 

shipping and storage of the samples will be detailed in Study Plan. 

Following preparation of composite samples of benthic macroinvertebrates, two subsamples of benthos 

shall be prepared for analysis of lipid content. Lipid content shall be analyzed using procedures 

equivalent to Powell and Woodburn (2009) Characterization of Fish and Benthic Macroinvertebrates, p. 

11 (see Annex 4).

Collection of fish samples for analysis of D4: 

On the day of field sampling at a given site, fish will be collected and sorted by species. Two species of 

fish will be selected that were collected in sufficient numbers or biomass such that composite samples 

can be prepared for the analysis of D4. For each of two species, two (n=2) composited samples shall be 

prepared in accordance with the sample collection procedures set out in the site-specific Sampling Plan. 

The sample collection procedures will be based, as appropriate, on USEPA (2003b, Section 7.0

Procedures) and Powell and Woodburn (2009, Sample Collection, p. 8, Analysis of Cyclic Volatile 

Methylsiloxane (cVMS) Materials section, Extraction of Fish and Benthic Macroinvertebrates subsection, p. 13, and Quality Control, p. 14)) (see Annexes 4-5). Sample collection methods as well as 

specific requirements for sample container preparation, sample handling, sample preservation, shipping 

and storage of the samples will be detailed in the site-specific Sampling Plans. 

During each sampling event at a given site, two field blanks and two field spikes will be prepared as 

follows. Serving as field blanks, two reference fresh ground fish samples having very low or nondetectable concentrations of cVMS materials shall be taken into the field and processed like a field 

sample. Rainbow trout purchased from a commercial fish hatchery will be ground into a homogenous 

mixture. Samples of the ground fish will be prepared in the laboratory prior to sample collection and 

taken to the field. The reference fish samples will be used to evaluate contamination bias resulting from 

collection, processing, transport, storage, and analysis of samples. Two of the ground fish samples shall 

be spiked in the field with a known amount of D4. The field spiked samples of ground fish will be used

to evaluate loss of cVMS during collection, processing, transport, storage, and analysis of samples, and 

to evaluate analytical bias. Specific requirements for preparation, processing, and analysis of field 

blanks, field spikes, and duplicate samples shall be detailed in Study Plan and the Quality Assurance 

Project Plan (QAPP).

Following preparation of composite samples of two fish species, two subsamples of each fish species 

shall be prepared for analysis of lipid content. Lipid content shall be analyzed using procedures 

equivalent to Powell and Woodburn (2009) Characterization of Fish and Benthic Macroinvertebrates, p. 

11 (see Annex 4).
Testing Standard for the Collection, Sample Handling, Transfer and Analysis of Biota Samples for D4

August 16, 2013

Silicones Environmental, Health and Safety Center 4

Analysis of Biota Samples for D4: 

All biota samples shall be analyzed for D4 at a commercial testing laboratory. The commercial laboratory 

will validate a method for the analysis of D4 in biota samples that shall be adapted from the analytical 

method specified in Powell and Woodburn (2009) (see Annex 4). The specific level of detection shall be 

based on the Practical Quantitation Limit (PQL) (USEPA 1985 and APHA 1999, Part 1030C, see Annexes 6 

and 7). The PQL is in turn based on the matrix-specific Method Detection Limit (MDL). These terms are 

defined as follows:

Practical Quantitation Limit (PQL): Minimum concentration of a constituent in a sample that can be 

measured among laboratories within specified limits during routine laboratory operations. The PQL 

is proposed because different laboratories may produce different MDLs even though they may be 

using the same analytical procedures, instruments and sample matrices. The PQL is typically defined 

as 5 to 10 times the MDL.

Method Detection Limit (MDL): Concentration of a constituent in a sample, that when processed 

through the complete analytical method, can be measured and reported with 99% probability that it 

is greater than 0 or statistically different from a blank. The MDL is a measure of a methods ability to 

quantify a constituent in a sample matrix.

The method for analyzing D4 in biota reported by Powell and Woodburn (2009) is deemed the 

appropriate method for transferring to a commercial testing laboratory and is capable of achieving an 

MDL of at least 3.3 ng/g (wet weight, ww) for biota. For use at the commercial laboratory, the targeted 

PQL for D4 in all biota shall be calculated as 5 times the Powell and Woodburn (2009) MDL of 3.3 ng/g, 

ww. Therefore, when rounded to one significant figure, the PQL for D4 in biota shall be 20 ng/g,ww.

Compliance with Good Laboratory Practices:

Sample collection, handling, and transfer activities should be conducted in accordance with GLP 

requirements, as applicable. The analytical laboratory conducting the analyses of D4 shall be a GLP 

certified facility and all analytical work shall be conducted in accordance with GLP requirements. The 

QAPP shall be prepared according to USEPA guidance and shall include information on all field and 

laboratory quality control measures to be followed. 

Annex 1 – USEPA 1991. Technical Support Document for Water Quality-based Toxics Control, Section 

4.4: Mixing Zone Analyses. EPA 505-290-001. U.S. Environmental Protection Agency, Office of Water, 

Washington, DC.

Annex 2 – USEPA 2006. Compilation of EPA Mixing Zone Documents, Mixing Zone Analyses. EPA 823-R06-003, July, 2006.
Testing Standard for the Collection, Sample Handling, Transfer and Analysis of Biota Samples for D4

August 16, 2013

Silicones Environmental, Health and Safety Center 5

Annex 3 – USEPA 2003a. Benthic Macroinvertebrate Sampling and Processing, Modeling Documents 

section, pp.9-13. Technical Standard Operating Procedure SOP#EH-04, September, 2003. 

Annex 4 – Powell DE, Woodburn KB. 2009. Trophic Dilution of Cyclic Volatile Methylsiloxane (cVMS) 

Materials in a Temperate Freshwater Lake. Dow Corning Corporation, HES Study No. 10771-108.

Annex 5 – USEPA 2003b. Fish Collection by Seining or Electrofishing. Technical Standard Operating 

Procedure SOP#EH-04, September, 2003.

Annex 6 – USEPA 1985. National primary water drinking standards: synthetic organics, inorganics, and 

bacteriologicals. 40 CFR Part 141; Federal Register 50: No. 219, November 13. 1985.

Annex 7 – APHA 1999. Standard Methods for the Examination of Water and Wastewater, Part 1030 C. 

Method Detection Level, Section 2 Determining Detection Levels. American Public Health Association, 

American Water Works Association, Water Environment Federation.