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Water: Beach Grants

National Beach Guidance and Required Performance Criteria - Appendix 4B2: Sample Collection

This appendix provides supplemental discussions, examples, and additional references that may be helpful to beach program managers. It does not create additional requirements beyond those in the main guidance document.

  1. Sample Containers
  2. Sampling Method
  3. Sample Handling
  4. References

Sample Containers

The sample bottles used to collect water for bacterial density analyses should be able to withstand sterilizing conditions and the solvent action of water. USEPA (1978) suggested wide-mouth borosilicate glass bottles with screw caps or ground-glass stoppers; however, glass bottles can break, causing loss of the sample. Heat-resistant polypropylene bottles may be used if they can be sterilized without producing toxic materials when autoclaved.

Sample bottles should be at least 125-milliliter (mL) volume for adequate sampling and for good mixing. Bottles of 250-mL, 500-mL, and 1,000-mL volume are often used for multiple analyses, such as when determining the density of two or more bacterial indicators. Discard bottles that have chips, cracks, or etched surfaces. Bottle closures should be watertight. Before use, bottles and closures should be cleaned with detergent and hot water, followed by a hot water rinse to remove all traces of detergent. Then rinse three times with laboratory-pure water.

Autoclave glass or heat-resistant polypropylene bottles at 121 C for 15 minutes. Alternatively, dry glassware may be sterilized in a hot air oven at 170 C for not less than 2 hours. Ethylene oxide gas sterilization should be acceptable for plastic containers that are not heat-resistant. Sample bottles should be stored overnight before they are used to allow the last traces of gas to dissipate.

Commercially available sterile plastic sampling bags (Whirl-pak) are a practical substitute for polypropylene or glass sample bottles when sampling soil or sediment. The bags are sealed by the manufacturer and opened only at the time of sampling.

If water samples are being collected for the determination of other environmental parameters (e.g., temperature, salinity, turbidity, dissolved oxygen), nonsterile containers may be used. It is important that the sterile and nonsterile containers are clearly labeled and used for the particular sample for which they were intended.

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Sampling Method

A grab sample of water is obtained using a sample bottle that has been prepared as described above. The basic steps for this procedure, derived from Bordner et al. (1978) and IITF (1999), are as follows.

  1. Identify the sampling site on a chain of custody tag, if required, or on the bottle label and on a field log sheet.
  2. Remove the bottle covering and closure just before obtaining each sample and protect them from contamination. Be careful not to touch the inside of the bottle itself or the inside of the cover.
  3. The first sample to be prepared is the trip or field blank (at least one per sampling day for routine sampling is recommended). Open one of the sampling bottles and fill it with 100 mL of sterile buffered dilution solution (see EPA Method 1103.1) when collecting freshwater, estuarine, or marine water samples. Cap the bottle and place it in a cooler.
  4. To collect the surface water samples, carefully move to the first sampling location. If wading in the water, try to avoid kicking up bottom material at the sampling station. The sampler should be positioned downstream of any water current to take the sample from the incoming flow.
  5. Open a sampling bottle and grasp it at the base with one hand and plunge the bottle mouth downward into the water to avoid introducing surface scum. Position the mouth of the bottle into the current away from the hand of the sampler and away from the side of the sampling platform or boat. The sampling depth should be 15 to 30 centimeters (6 to 12 inches) below the water surface, depending on the depth from which the sample must be taken. If the waterbody is static, an artificial current can be created by moving the bottle horizontally with the direction of the bottle pointed away from the sampler. Tip the bottle slightly upward to allow air to exit and the bottle to fill.
  6. Remove the bottle from the waterbody.
  7. Pour out a small portion of the sample to allow an air space of 2.5 centimeters (1 to 2 inches) above each sample for proper mixing of the sample before analysis.
  8. Tightly close the stopper and label the bottle.
  9. Enter specific details to identify the sample on a permanent label. Take care in transcribing sampling information to the label. The label should be clean, waterproof, nonsmearing, and large enough for the necessary information. The label must be securely attached to the sample bottle but removable when necessary. Preprinting standard information on the label can save time in the field. The marking pen or other device must be nonsmearing and maintain a permanent legible mark.
  10. Complete a field record for each sample to record the full details on sampling and other pertinent remarks, such as flooding, rain, or extreme temperature, that are relevant to interpretation of the results. This record also provides a back-up record of sample identification.
  11. Place the samples in a suitable container and transport them to the laboratory as soon as possible. Adhering to sample preservation and holding time limits is critical to the production of valid data. Bacteriological samples should be iced or refrigerated at 1 to 4 C during transit to the laboratory. Use insulated containers such as plastic or styrofoam coolers, if possible, to ensure proper maintenance of storage temperature. Take care to ensure that sample bottles are not totally immersed in water during transit or storage. Examine samples as soon as possible after collection. Do not hold samples longer than 6 hours between collection and initiation of analysis (USEPA, 2000). Do not analyze samples that exceed holding time limits.
  12. Collect water samples for analyses of other parameters in separate appropriate containers at the same time and perform analyses as specified in the particular methods.
  13. After collecting samples from a station, wash hands and arms with alcohol wipes, a disinfectant lotion, or soap and water, and dry to reduce exposure to potentially harmful bacteria or other microorganisms.

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Sample Handling

In cases where an agency must demonstrate the reliability of its evidence in legal cases involving pollution, it is important to document the chain of possession and custody of samples that are offered for evidence or that form the basis of analytical results introduced into evidence (Bordner et al., 1978). Although the analytical results of the water samples collected at a swimming beach are being used to make a decision for the protection of human health, a decision to close the beach might be unpopular with local businesses and could be contested. It is thus important that the agency collecting the samples and the laboratory performing the analysis prepare written procedures to be followed whenever evidence samples are collected, transferred, stored, analyzed, or destroyed. These are known as "chain of custody" (COC) procedures.

The sampling agency should have procedures to ensure the custody and integrity of the samples beginning at the time of sampling and continuing through transport and sample receipt. The laboratory should have procedures for sample receipt, preparation, analysis and storage, data generation and reporting, and sample disposal.

A COC form filled out by the person conducting the sampling should provide information such as the following: sampling location (site ID), time of collection, date of collection, time of near or high tide, air temperature, water temperature, rainfall history, collector's name and signature, agency, and other notes or comments. A Chain-of-Custody Review List and a Sample Handling, Preparation, and Analysis List are provided at the end of this appendix.

Samples are usually transported to the laboratory by the person collecting the sample or picked up by laboratory personnel. Because of the 6-hour holding time limitation, the laboratory should be conveniently located near the sampling site and should be notified a few days in advance of the sampling effort so that it will be ready to process the samples promptly. COC procedures should be followed at the laboratory for all samples. Laboratory personnel receiving samples should do the following:

  1. Check the shipping container for damage and a custody seal. Note whether the custody seal is intact and record any anomalies on the sample log-in form.
  2. Open the container and inspect the sample containers, noting any damage or breakage. Immediately take the temperature of the samples. Place a calibrated thermometer or temperature probe in the cooler in a representative location (not directly touching any ice or cold packs and not inside a sample bottle). Record the temperature on the sample log-in form and the COC form enclosed with the sample.
  3. Remove the individual containers from the shipping container and inspect each one for damage, leakage, or any other problem. Note the condition of each container, the date received, the project number, the batch number, and the airbill or shipping identification number on the sample log-in form and the COC form.
  4. Compare each sample container to those listed on the COC form to ascertain whether all the samples are present and whether all the labels on the sample containers match those on the COC form.
  5. If no COC form accompanies the samples, complete a COC form and confirm all sample information with the agency that collected the samples. Document any contact with the agency regarding problems or confirmation on the sample log-in and COC forms.
  6. Notify the laboratory manager if any problems with the samples are noted. Sign and date the COC form upon completion of the sample inspection.
  7. Assign each sample a sample ID code. For example, the sample ID code should include a sequential log-in number, a sample type code (e.g., U for upstream, S for site, L for laboratory), a code to identify the collecting agency, the sampling date, and the analysis required. Assign replicate samples from the same site the same code with a suffix such as -A,- B, or -C to indicate their replicate status.
  8. Record each sample's code on the sample log-in form, the COC form, and the corresponding sample container. Indicate on the form where the samples will be held (e.g., which room in the laboratory). When samples are removed for final disposition, the removal should be documented on the sample log-in form.
  9. Record additional information on the sample log-in form, including the collecting agency contact, sample analyses required, and due dates of analyses.
  10. Store samples not used immediately at 4°C.
Table 1. Chain-of-Custody Review List
Sample custodian designated
Name of sample custodian
Sample custodian's procedures and responsibilities documented
Standard operating procedures (SOPs) developed for receipt of samples
Where are the SOPs documented (laboratory manual, written instructions...)?
Receipt of chain-of-custody record(s) with samples documented
Nonreceipt of chain-of-custody record(s) with samples documented
Integrity of the shipping container(s) documented
Where is security documented?
Lack of integrity of the shipping container(s) documented
Where is nonsecurity documented?
Agreement between chain-of-custody records and sample tags verified and documented
Source of verification and location of documentation
Sample tag numbers recorded by the sample custodian
Where are they located?
Samples stored in a secure area
Where are they stored?
Sample identification maintained
Sample extract (or inorganics concentrate) identification
Samples maintained. How?

Table 2. Sample Handling, Preparation, and Analysis List
Category Task
Field Logs Project name/ID and location
Sampling personnel identified
Geological observations
Atmospheric conditions
Field measurements
Sample dates, times, and locations
Sample identifications noted
Sample matrix identified
Sample descriptions (e.g., odors, colors)
Number of samples taken per location
Sampling method/equipment
Description of any QC samples
Deviations from the sampling plan
Difficulties or unusual circumstances
Chain-of-Custody Records Project name/ID and location
Sample custodian's procedures and responsibilities documented
Sample custodians' signatures verified and on file
Date and time of each transfer
Carrier ID number
Integrity of shipping container and seals verified
Standard operating procedures (SOPs) for receipt on file
Samples stored in same area
Holding time protocol verified
SOPs for sample preservation on file
Identification of proposed analytical method verified
Proposed analytical method documentation verified
QA plan for proposed analytical method on file
Sample Labels Sample ID
Date and time of collection
Sampler's signature
Characteristic or parameter investigated
Preservative used
Sample Receipt Log Date and time of receipt
Sample collection date
Client sample ID
Number of samples
Sample matrices
Requested analysis, including method number(s)
Signature of the sample custodian or designee
Sampling kit code (if applicable)
Sampling condition
Chain-of-custody violations and identities
Sample Preparation Logs Parameter/analyte of investigation
Method number
Date and time of preparation
Analyst's initials or signature
Initial sample volume or weight
Final sample volume
Concentration and amount of spiking solutions used
QC samples included with the sample batch
ID for reagents, standards, and spiking solutions used
Sample Analysis Logs Parameter/analyte of investigation
Method number/reference
Date and time of analysis
Analyst's initials or signature
Laboratory sample ID
Sample aliquot
Dilution factors and final sample volumes (if applicable)
Absorbance values, peak heights, or initial concentrations reading
Final analyte concentration
Calibration data (if applicable)
Correlation coefficient (including parameters)
Calculations of key quantities available
Comments on interferences or unusual observations
QC information, including percent recovery
Instrument Run Logs Name/type of instrument
Instrument manufacturer and model number
Serial number
Date received and date placed in service
Instrument ID assigned by the laboratory (if used)
Service contract information, including service representative details
Description of each maintenance or repair activity performed
Date and time of each maintenance or repair activity
Initials of maintenance or repair technicians
Chemical/Standard Receipt Logs Laboratory control number
Date of receipt
Initials or signature of person receiving chemical
Chemical name and catalog number
Vendor name and log number
Concentration or purity of standard
Expiration date
Standards/Reagent Preparation Log Date of preparation
Initials of analyst preparing the standard solution or reagent
Concentration or purity of standard or reagent
Volume or weight of the stock solution
Final volume of the solution being prepared
Laboratory ID/control number assigned to the new solution
Name of standard reagent
Standardization of reagents, titrants, etc. (if applicable)
Expiration date

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  1. Bordner, R., J.A. Winter, and P.V. Scarpino, eds. 1978. Microbiological Methods for Monitoring the Environment, Water and Wastes. EPA-600/8-78-017. U.S. Environmental Protection Agency, Washington, DC.
  2. IITF. 1999. Standard Operating Procedure for Recreational Water Collection and Analysis of E. coli on Streams, Rivers, Lakes and Wastewater. Indiana Interagency Task Force on E. coli. LaPorte County Health Department, LaPorte, IN.
  3. USEPA. 1978. Microbiological Methods for Monitoring the Environment, Water and Wastes. EPA 600/8-78-017. U.S. Environmental Protection Agency, Washington, DC.
  4. USEPA. 2000. Improved Enumeration Methods for the Recreational Water Quality Indicators: Enterococci and Escherichia coli. EPA 821/R-97-004. U.S. Environmental Protection Agency, Office of Science and Technology, Washington, DC.

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