Testing Standard for the Collection, Sample Handling, Transfer and Analysis of Biota Samples for D4 August 1 6 , 2013

Silicones Environmental, Health and Safety Center 1 Synopsis:

This testing standard provide s 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 Sampl ing Plans for the collection and analysis of bio ta samples, which will be included in a Study Plan for the Testing Program.

Requirements for s ampling biota :

Per Appendix 1, Environmental Testing at D4 Direct Discharge Sites
, Appendix 2, Environmental T esting 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 sh ould 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 th e 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 e stablished. 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, establish ment of the deposition zone by field reconnaissance shall b e detailed in the site

- specific Sampling Plans.Testing Standard for the Collection, Sample Handling, Transfer and Analysis of Biota Samples for D4 August 1 6 , 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 o f 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 zo ne 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 analys is 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 b iota 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 lubricant s, a nd may be components of antifoams widely used in w aste 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 fie ld. For example, field crew shall refrain from using any personal care products (e.g. , sun

- block, sun

screen, hand lotion, antiperspirants, etc.) while preparing for or conducting any field activities to minimize the possibility for contamination with cVM S 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 b e 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

or ganisms shall be prepared. The sampl e collection procedures will be based, as appropriate, on techniques described in USEPA ( 2003 a , Sections 5.2 Sampling for Community Samples and 5.3 Sampling for Analytical Measurements ) and Powell and Woodburn (2009 , Sample Collection, p. 9
, Analysis ofTesting Standard for the Collection, Sample Handling, Transfer and Analysis of Biota Samples for D4 August 1 6 , 2013

Silicones Environmental, Health and Safety Center 3 Cyclic Volatile Methylsiloxane (cVMS) Materials section , Extraction of Fish and Benthic Macroinvertebrates sub

- section , p. 1 3, and Quality Control, p. 14 ) (see Annexes 3

- 4 ). Sample collection procedures as well as spe cific 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 prep ared 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 ea ch of two species, t wo (n=2) composited sa mples shall be prepared i n accordance with the sample collection procedures set out in the site

- specific Sampling Plan. The sampl e collection procedures will be based, as appropriate, on USEPA ( 2003 b , 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 sub

section , p. 1 3, and Quality Control, p. 14 ) ) (see Annexes 4

- 5 ). S ample coll ection 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 giv en site, two field blank s and two field spike s will be prepared as follows . Serving as field blank s , two reference fresh ground fish sample s having very low or non

detectable 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 t o the field. The reference fish sample s 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 fi eld blanks, field spikes, and duplicate samples sh all 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 analysi s 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 1 6 , 2013

Silicones Environmental, Health and Safety Center 4 Analysis of Biota Samples for D4:

All biota samples shall be ana lyzed 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 analy tical method specified in Powell and Woodburn (2009) (see Annex 4 )

. The specific l evel of detection shall be based on the Practical Quantitation Limit (PQL) (USEPA 1985 and APHA 1999, Part 1030C, see Annex es 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 di fferent 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 process ed 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 met hod 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 a n MDL of at least 3.3 ng/g (wet weight , ww ) for biota

. For use at the com mercial laboratory, the targeted PQL for D4 in all biota shall be calculated as 5 times the Powell and Woodburn (20 09 ) 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 Goo d 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 analytic al 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.

A nnex 1

– U SEPA 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.

A nnex 2

– USEPA 2006. Compilation of EPA Mixing Zone Documents
, Mixing Zone Analyses . EPA 823

- R

06

- 003, July, 2006.Testing Standard for the Collection, Sample Handling, Transfer and Analysis of Biota Samples for D4 August 1 6 , 2013

Silicones Environmental, Health and Safety Center 5 Annex 3

– USEP A 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. 200 9. Trophic Dilution of Cyclic Volatile Methylsiloxane (cVMS) Materials in a Temperate Freshwater Lake. Dow Corning Corporation, HES Study No. 10771

- 108.

Annex 5

– USEPA 200 3b. Fish Collection by Seining or Electrofishing. Technical Standard Operating
Proce dure 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 Meth ods 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.