       Supporting Statement for an Information Collection Request (ICR)
                    Under the Paperwork Reduction Act (PRA)
EXECUTIVE SUMMARY
Identification of the Information Collection  -  Title and Numbers
	Title: 	Labeling Requirements for Certain Minimum Risk Pesticides under FIFRA Section 25(b)
	EPA ICR No.: 2475.04
	OMB Control No.: 2070-0187
	Docket ID No.: EPA-HQ-OPP-2021-0346
Abstract
This information collection request documents the Paperwork Reduction Act (PRA) burden for the labeling requirements for certain minimum risk pesticide products exempt from Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) registration under 40 CFR 152.25(f). These requirements were updated in the final rule entitled: Pesticides; Revisions to Minimum Risk Exemption (80 FR 80653; December 28, 2015).
Under 40 CFR 152.25(f), EPA has exempted from the requirement of FIFRA registration certain pesticide products if they are composed of specified ingredients and labeled accordingly. EPA created the exemption for minimum risk pesticides to eliminate the need for industry or business to expend significant resources to apply for and maintain regulated products that are deemed to be of minimum risk to human health and the environment. In addition, exempting such products freed Agency resources to focus on evaluating formulations whose toxicity was less well characterized, or was of higher toxicity.
The 2015 Final Rule reorganized the ingredients lists and added specific chemical identifiers to clarify to manufacturers, the public, and Federal, state, and tribal inspectors the specific chemical substances that are permitted in minimum risk pesticide products. EPA also modified the label requirements to require the use of specific label display names of ingredients and to require producer contact information on the label. The primary goal of this rulemaking was to clarify the conditions of exemption for minimum risk pesticides by clarifying the specific ingredients that are permitted in minimum risk pesticide products and to provide company contact information on the label. The previous version of this ICR covered the paperwork burdens associated with existing products updating their labels to comply with the new requirements during the 2015 Final Rule's compliance period. EPA anticipates that those burdens have been realized, and is now accounting for the potential burden for new products coming into the market.
Summary Total Burden and Costs

                                     TOTAL

                                   Responses
                                     Hours
                                     Costs
Annual Labeling Activities
                                                                             87
                                                                          478.5
                                                                     $61,018.45
Total Response Burden
                                                                            260
                                                                        1,435.5
                                                                    $183,055.35
Agency Burden Estimate

                                                                          00.00
                                                                          00.00

Supporting Statement
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Explain the circumstances that make the collection of information necessary. Identify any legal or administrative requirements that necessitate the collection. Attach a copy of the appropriate section of each statute and regulation mandating or authorizing the collection of information.
Authorizing legislation is contained in section 3 and 25 of FIFRA, as amended. Requirements for labels of minimum risk pesticide products are described in 40 CFR 152.25(f) (See attachments A, B, and C).
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Indicate how, by whom, and for what purpose the information is to be used.  Except for a new collection, indicate the actual use the Agency has made of the information received from the current collection
Under FIFRA 25(b)(2), EPA may exempt from the requirements of FIFRA any pesticide that is "of a character unnecessary to be subject to [FIFRA]." Pursuant to this authority, in March 1996, EPA promulgated 40 CFR 152.25(g), which exempted from FIFRA any pesticide products consisting solely of specified ingredients that EPA judged to pose minimum risk to humans and the environment (61 FR 8876, March 6, 1996). This provision was later redesignated as 152.25(f) (66 FR 64759, December 14, 2001). Unlike producers of registered pesticides, producers of products exempted under 152.25(f) do not register their products with EPA, pay registration fees, or report production to EPA.
This exemption is in contrast to a typical FIFRA section 3 registration of a pesticide. A section 3 registration is a scientific, legal, and administrative process through which EPA examines the ingredients of the pesticide; the particular site or crop on which it is to be used; the amount, frequency and timing of its use; and storage and disposal practices. In evaluating a pesticide registration application, EPA assesses a wide variety of potential human health and environmental effects associated with use of the product. The producer of the pesticide must provide data to EPA, using tests done according to either EPA guidelines or other methods determined acceptable by EPA on a case-by-case basis. The data from these tests are used to determine whether a pesticide has the potential to cause adverse effects on humans, wildlife, fish, and plants, including endangered species and non-target organisms, as well as possible contamination of surface water or groundwater from leaching, runoff and spray drift. Potential human risks include short-term toxicity and long-term effects such as cancer and reproductive system disorders. EPA also must approve the language that appears on each pesticide label. A pesticide product can only be used according to the directions on the label or labeling accompanying it at the time of sale, through its use and disposal. The labeling is the primary enforcement mechanism for Federal, state, and tribal authorities.
Since minimum risk pesticide products are not registered by EPA, the product information associated with the pesticide registration process under section 3 of FIFRA are never submitted to EPA. However, approximately 39 states and the District of Columbia require products that are exempt from FIFRA requirements under 152.25(f) to obtain a state-registration. Generally, state registration of a federally-registered pesticide relies heavily on the previous Federal review of the product's toxicity, use patterns, and label. In contrast, given that minimum risk pesticides are exempt from Federal registration under FIFRA, the numerous states that do regulate these products use review criteria that vary from state to state. In some states, manufacturers of minimum risk products are only required to pay a registration fee; in others, there is a label review, which can include a review of the ingredients used in the product; and a few require Material Safety Data Sheets and data on product efficacy.
Thus, labeling requirements are the key component of the minimum risk exemption since this is the only information that enforcement authorities have to assess whether or not the product meets the exemption requirements. While EPA does not review these products, and therefore a Federal label review is not conducted, to maintain exemption status, an exempt product's label must meet certain criteria. The methods for displaying active and inert ingredient information are detailed in the exemption: labels must include the EPA required "label display name" and the percentage (by weight) of active ingredients and list all inert ingredients by their label display name. The label display name for a chemical is name that is codified in the minimum risk regulations at 40 CFR 152.25(f)(1) and (2). Other names for the chemical/substance may exist, but the ingredient statement should only use the EPA label display name. The label information documented in this renewal ICR accounts for the burden of labeling new products entering the market. These labels provide important regulatory information for the Federal, state, and tribal authorities that regulate or enforce minimum risk products.
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Describe whether, and to what extent, the collection of information involves the use of automated, electronic, mechanical, or other technological collection techniques or other forms of information technology, e.g., permitting electronic submission of responses, and the basis for the decision for adopting this means of collection. Also describe any consideration of using information technology to reduce burden.
Not applicable.
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Describe efforts to identify duplication. Show specifically why any similar information already available cannot be used or modified for use for the purposes described in Item 2 above.
Duplication will not occur in this program, as the labeling requirements are unique to each minimum risk product. The exemption also does not require submission of information to EPA. The exemption standardizes some of the information on product labels, which reduces the burden on industry by creating labels for the same product registered for use in different states
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If the collection of information impacts small businesses or other small entities, describe the methods used to minimize burden.
EPA created the exemption for minimum risk pesticides to eliminate the need to expend significant resources to regulate products that were deemed to be of minimum risk to human health and the environment. Since these minimum risk products do not have to be registered at the Federal level, significant cost savings are available for small business to benefit from this exemption.
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Describe the consequence to Federal program or policy activities if the collection is not conducted or is conducted less frequently, as well as any technical or legal obstacles to reducing burden.
The frequency of the collection (3 years) cannot be reduced. This information collection activity is a one-time collection for new minimum risk products entering into the market. This collection provides an accounting of the burden for any new products entering into the market that must comply with the labeling requirements in the minimum risk exemption.
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Explain any special circumstances that require the collection to be conducted in a manner:
 requiring respondents to report information to the agency more often than quarterly;
 requiring respondents to prepare a written response to a collection of information in fewer than 30 days after receipt of it;
 requiring respondents to submit more than an original and two copies of any document;
 requiring respondents to retain records, other than health, medical, government contract, grant-in-aid, or tax records, for more than three years;
 in connection with a statistical survey, that is not designed to produce valid and reliable results that can be generalized to the universe of study;
 requiring the use of a statistical data classification that has not been reviewed and approved by OMB;
 that includes a pledge of confidentiality that is not supported by authority established in statute or regulation, that is not supported by disclosure and data security policies that are consistent with the pledge, or which unnecessarily impedes sharing of data with other agencies for compatible confidential use; or
 requiring respondents to submit proprietary trade secrets, or other confidential information unless the agency can demonstrate that it has instituted procedures to protect the information's confidentiality to the extent permitted by law.
Not applicable.
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If applicable, provide a copy and identify the date and page number of publication in the Federal Register of the agency's notice, required by 5 CFR 1320.8(d), soliciting comments on the information collection prior to submission to OMB. Summarize public comments received in response to that notice and describe actions taken in response to the comments. Specifically address comments received on cost and hour burden.

Describe efforts to consult with persons outside EPA to obtain their views on the availability of data, frequency of collection, the clarity of instructions and recordkeeping, disclosure, or reporting format (if any), and on the data elements to be recorded, disclosed, or reported.

Consultation with representatives of those from whom information is to be obtained or those who must compile records should occur at least once every 3 years - even if the collection of information activity is the same as in prior periods. There may be circumstances that may preclude consultation in a specific situation. These circumstances should be explained.
Pursuant to 5 CFR 1320.8(d) in proposing to renew this ICR, EPA will publish a notice in the Federal Register that will provide a 60-day public comment period. The comments received and any additional public comment for this action will be posted in the docket.
As required under 5 CFR 1320.8(d)(1), in addition to the public notice EPA staff will contact appropriate stakeholders and ask them for their assessment of the regulatory burden estimates expressed by the Agency in this ICR. A summary of these consultations will be provided in the supporting statement during the second comment period. The full consultation will be reserved in Attachment D.
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Explain any decision to provide any payment or gift to respondents, other than remuneration of contractors or grantees.
Not applicable
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Describe any assurance of confidentiality provided to respondents and the basis for the assurance in statute, regulation, or agency policy. If the collection requires a systems of records notice (SORN) or privacy impact assessment (PIA), those should be cited and described here.
EPA's policy is that all comments received will be included in the docket without change, including any personal information provided, unless the comment includes profanity, threats, information claimed to be Confidential Business Information (CBI) or other information whose disclosure is restricted by statute.
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Provide additional justification for any questions of a sensitive nature, such as sexual behavior and attitudes, religious beliefs, and other matters that are commonly considered private. This justification should include the reasons why the agency considers the questions necessary, the specific uses to be made of the information, the explanation to be given to persons from whom the information is requested, and any steps to be taken to obtain their consent.
No information of a sensitive or private nature is requested in conjunction with these information collection activities, and these information collection activities comply with the provisions of the Privacy Act of 1974 and OMB Circular A-108.
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Provide estimates of the hour burden of the collection of information. The statement should:
 Indicate the number of respondents, frequency of response, annual hour burden, and an explanation of how the burden was estimated. Unless directed to do so, agencies should not conduct special surveys to obtain information on which to base hour burden estimates. Consultation with a sample (fewer than 10) of potential respondents is desirable. If the hour burden on respondents is expected to vary widely because of differences in activity, size, or complexity, show the range of estimated hour burden, and explain the reasons for the variance. Generally, estimates should not include burden hours for customary and usual business practices.
   b) 	If this request for approval covers more than one form, provide separate hour burden estimates for each form and aggregate the hour burdens.
   c) 	Provide estimates of annualized cost to respondents for the hour burdens for collections of information, identifying and using appropriate wage rate categories. The cost of contracting out or paying outside parties for information collection activities should not be included here. Instead, this cost should be included under `Annual Cost to Federal Government'.
Minimum risk pesticide products are exempt from federal registration requirements, and manufacturers of these products do not submit any data, forms, or labels to EPA. They are also not required to conduct annual reporting or recordkeeping. The requirements for minimum risk pesticide products are limited to what active and inert ingredients they may contain, and specific information that must be on product labels. However, to help companies comply with the labeling requirements, EPA provides and periodically updates web guidance applicable to minimum risk products.
There is no change in the total estimated respondent burden compared with the ICR currently approved by OMB. This is based on the assumption that the estimated number of new products entering the market is the same as that used in the previous ICR, that is, 87 per year, with estimated burden hours of 478.5 hours per year. However, in this renewal, burden costs are updated with the 2019 wage rates.
The total burden hours per response (5.5 hours) has not changed for this ICR renewal and the total response burden estimate has remained the same at 1,435.5 hours. With updated wage rates, the total labor costs increased from $156,606.53 to $183,055.35.
Over the next three years, EPA estimates that there will be approximately 49 companies selling approximately 170 new products that meet the requirements of the minimum risk pesticide exemption in the U.S. These estimates are derived primarily from estimates provided by three states (Indiana, Maine, and New Mexico) who register minimum risk pesticides in their states. Although minimum risk products are exempt from registration by EPA, most states require some form of registration for these pesticide products. Additionally, several of these states provide their pesticide registration lists online (including minimum risk pesticides) and are available to the public.
However, many products have more than one size or type of package. Each is referred to as a stock keeping unit (SKU). Each SKU would have to be labeled to comply with the new requirements established in 2015. EPA has estimated that there are 1.53 SKUs per product, for a total number of 260 products that would have to be labeled according the exemption's requirements. Therefore, approximately 87 new products are expected to enter the market per year over the next 3 years.
The total cost for the labeling activities over 3 years is estimated to cost $156,607, for a one-time burden. The annual burden is estimated to cost $52,202 per year. Cost rates have been indexed to May 2019 dollars.
Agency economists revised the estimated wages, benefits, and overhead for all labor categories for affected industries, state government, and EPA employees based on publicly available data from the US Bureau of Labor Statistics. The formulas used to estimate the labor rates and formulas used to derive the fully loaded rates and overhead costs for this ICR are listed in Attachment E.
Table 1. Wage Rates
Methodology
The methodology uses data on each sector and labor type for an Unloaded wage rate (hourly wage rate) and calculates the Loaded wage rate (unloaded wage rate + benefits), and the Fully loaded wage rate (loaded wage rate + overhead). Fully loaded wage rates are used to calculate respondent costs. This ICR uses 2019 wage data.

Unloaded Wage Rate
Wages are estimated for labor types (management, technical, and clerical) within applicable sectors. The Agency uses average wage data for the relevant sectors available in the National Industry-Specific Occupational Employment and Wage Estimates from the Bureau of Labor Statistics (BLS) at http://www.bls.gov/oes/current/oes_nat.htm.

Sectors
The specific North American Industry Classification System (NAICS) code and website for each sector is included in that sector's wage rate table (see Attachment E). Within each sector, the wage data are provided by Standard Occupational Classification (SOC). The SOC system is used by Federal statistical agencies to classify workers into occupational categories for the purpose of collecting, calculating, or disseminating data (see http://www.bls.gov/oes/current/oes_stru.htm).

Loaded Wage Rate
Unless stated otherwise, all benefits represent 46.4% of unloaded wage rates, based on benefits for all civilian non-farm workers, from http://www.bls.gov/news.release/ecec.t01.htm. However, if other sectors are listed for which 46.4% is not applicable, the applicable percentage will be stated.

Fully Loaded Wage Rate
We multiply the loaded wage rate by 50% (EPA guidelines 20-70%) to get overhead costs.

The following table presents the estimated burden and cost estimates per label to comply with the information collection activities associated with the rulemaking:
Table 2: Estimated Burden/Cost per Label (Minimum Risk Exemption - New Product Labeling; No Registration)
Collection Activities
                                 Burden Hours
                                     Total

                                  Mgmt. $/hr
                                  Tech. $/hr
                                  Cler. $/hr
                                     Hours
                                    Costs $

                                   $145.34
                                    $76.35
                                    $49.61

Read Instructions
                                      3.5
                                      0.0
                                      0.0
                                                                            3.5
                                                                         508.67
Plan activities
                                      0.5
                                      0.0
                                      0.0
                                                                            0.5
                                                                          72.67
Gather/create information
                                      0.0
                                      1.5
                                      0.0
                                                                            1.5
                                                                         114.52
TOTAL
                                      4.0
                                      1.5
                                      0.0
                                                                            5.5
                                                                         695.86

Annual Burden & Costs: 	5.5 hours x 87 responses/year = 	478.5 hours/year
   (a) Management:	4.0 hours x $145.34 x 87 Responses = 	$ 50,576.67
   (b) Technical:	1.5 hours x $76.35 x 87 Responses =   	$   9,963.28
   (c) Clerical:	0 hours x $49.61 x 87 Responses =       	$               0
   	                                                 Total =      	$ 61,018.45
Total Burden & Costs:	5.5 hours x (87 responses/year x 3 years) = 	1,435.5 hours
   (a) Management:	4.0 hours x $145.34 x (87 responses/year x 3 years)  = 	$ 151,730.00
   (b) Technical:	1.5 hours x $76.35 x (87 responses/year x 3 years) =   	$   29,889.85
   (c) Clerical:	0 hours x $49.61 x (87 responses/year x 3 years) =       	$                 0
   	                                                		 Total =      	$ 183,055.35

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Provide an estimate for the total annual cost burden to respondents or recordkeepers resulting from the collection of information. (Do not include the cost of any hour burden already reflected on the burden worksheet).
 The cost estimate should be split into two components: (a) a total capital and start-up cost component (annualized over its expected useful life) and (b) a total operation and maintenance and purchase of services component. The estimates should take into account costs associated with generating, maintaining, and disclosing or providing the information. Include descriptions of methods used to estimate major cost factors including system and technology acquisition, expected useful life of capital equipment, the discount rate(s), and the time period over which costs will be incurred. Capital and start-up costs include, among other items, preparations for collecting information such as purchasing computers and software; monitoring, sampling, drilling and testing equipment; and record storage facilities.
 If cost estimates are expected to vary widely, agencies should present ranges of cost burdens and explain the reasons for the variance. The cost of purchasing or contracting out information collections services should be a part of this cost burden estimate. In developing cost burden estimates, agencies may consult with a sample of respondents (fewer than 10), utilize the 60-day pre-OMB submission public comment process and use existing economic or regulatory impact analysis associated with the rulemaking containing the information containing the information collection, as appropriate.
 Generally, estimates should not include purchases of equipment or services, or portions thereof, made: (1) prior to October 1, 1995, (2) to achieve regulatory compliance with requirements not associated with the information collection, (3) for reasons other than to provide information or keep records for the government, or (4) as part of customary and usual business or private practices.
There are no operational or maintenance costs associated with this collection.
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Provide estimates of annualized cost to the Federal government. Also, provide a description of the method used to estimate cost, which should include quantification of hours, operational expenses (such as equipment, overhead, printing, and support staff), and any other expense that would not have been incurred without this collection of information. Agencies may also aggregate cost estimates from Items 12, 13, and 14 in a single table.
There is no Agency burden related to registration of minimum risk pesticide products since they are exempt from Federal registration.
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Explain the reasons for any program changes or adjustments reported in hour or cost burden.
There is no change in the total estimated respondent burden compared with the ICR currently approved by OMB. This is based on the assumption that the estimated number of new products entering the market is the same as that used in the previous ICR, that is, 87 per year, with estimated burden hours of 478.5 hours per year. However, in this renewal, burden costs are updated with the 2019 wage rates.
The total burden hours per response (5.5 hours) has not changed for this ICR renewal and the total response burden estimate has remained the same at 1,435.5 hours. With updated wage rates, the total labor costs increased from $156,606.53 to $183,055.35.
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For collections whose results will be published, outline the plans for tabulation and publication. Address any complex analytical techniques that will be used. Provide the time schedule for the entire project, including beginning and ending dates of the collection of information, completion of report, publication dates, and other actions.
Not applicable.
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If seeking approval to not display the expiration date for OMB approval of the information collection, explain the reasons why display would be inappropriate.
Not applicable.
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Explain each exception to the certification statement identified in "Certification for Paperwork Reduction Act Submissions."
EPA does not request an exception to the certification of this information collection.
Supplemental Information
This collection of information is approved by OMB under the Paperwork Reduction Act, 44 U.S.C. 3501 et seq. (OMB Control No. 2070-0187). Responses to this collection of information are mandatory for certain persons, as specified at 40 CFR 152.25(f). An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB control number. The public reporting and recordkeeping burden for this collection of information is estimated to be 5.5 hours per response. Send comments on the Agency's need for this information, the accuracy of the provided burden estimates and any suggested methods for minimizing respondent burden to the Regulatory Support Division Director, U.S. Environmental Protection Agency (2821T), 1200 Pennsylvania Ave., NW, Washington, D.C. 20460. Include the OMB control number in any correspondence. Do not send the completed form to this address.
List of Attachments
The attachments listed below can be found in the docket for this ICR or by using the hyperlink that is provided in the list below. The docket for this ICR is accessible electronically through http://www.regulations.gov using Docket ID Number: EPA-HQ- OPP-2018-0139.
Attachment
Title
                                      A.
Title 7 Agriculture, §136a. Registration of Pesticides
                                      B.
Title 7: Agriculture, §136v Authority of States
                                      C.
Title 40: Protection of Environment, PART 152 Pesticide Registration and Classification Procedures, Subpart B -- Exemptions, § 152.25:
                                       D
[Reserved for Consultations]
                                      E.
Work Sheet Used to Calculate Pesticide Industry Labor Costs


STANDARD METHODS 9221 B. STANDARD TOTAL COLIFORM FERMENTATION TECHNIQUE JUNE 2003 DRAFT Draft 1 June 2003 STANDARD METHODS 9221 B. STANDARD TOTAL COLIFORM FERMENTATION TECHNIQUE Reprinted by Permission from the 20th Edition 1. Presumptive Phase Use lauryl tryptose broth in the presumptive portion of the multiple tube test. If the medium has been refrigerated after sterilization, incubate overnight at room temperature ( 20/ C) before use. Discard tubes showing growth and/ or bubbles.

a. Reagents and culture medium:

1) Lauryl tryptose broth:

Tryptose ................................................................................ 20.0 g Lactose ............................................................................... ..... 5.0 g Dipotassium hydrogen phosphate. K2HPO4 ........................... 2.75 g Potassium dihydrogen phosphate. KH2PO4 ........................... 2.75 g Sodium chloride. NaC1 ........................................................... 5.0 g Sodium lauryl sulfate .............................................................. 0.1 g Reagent grade water .............................................................. .. 1 L Add dehydrated ingredients to water, mix thoroughly, and heat to dissolve. pH should be 6.8 ± 0.2 after sterilization. Before sterilization, dispense sufficient medium, in fermentation tubes with an inverted vial, to cover inverted vial at least one half to two thirds after sterilization. Alternatively, omit inverted vial and add 0.01 g/ L bromcresol purple to presumptive medium to determine acid production. the indicator of a positive result in this part of the coliform test. Close tubes with metal or heat resistant plastic caps.

Make lauryl tryptose broth of such strength that adding 100 mL, 20 mL, or l0 mL portions of sample to medium will not reduce ingredient concentrations below those of the standard medium. Prepare in accordance with Table 9221: 1.

b. Procedure:

1) Arrange fermentation tubes in rows of five or ten tubes each in a test tube rack. The number of rows and the sample volumes selected depend upon the quality and character of the water to be examined. For potable water use five 20 mL portions, ten 10 mL portions, or a single bottle of 100 mL portion; for nonpotable water use five tubes per dilution ( of 10, 1, 0.1 mL, etc.).

In making dilutions and measuring diluted sample volumes, follow the precautions given in Section 9215B. 2. Use Figure 9215: 1 as a guide to preparing dilutions. Shake sample and dilutions vigorously about 25 times. Inoculate each tube in a set of five with replicate sample volumes ( in increasing decimal dilutions, if decimal quantities of the sample are used). Mix test portions in the medium by gentle agitation. Draft 2 June 2003 2) Incubate inoculated tubes or bottles at 35 ± 0.5C. After 24 ± 2 h swirl each tube or bottle gently and examine it for growth, gas, and acidic reaction ( shades of yellow color) and, if no gas or acidic reaction is evident, reincubate and reexamine at the end of 48 ± 3 h. Record presence or absence of growth, gas, and acid production. If the inner vial is omitted, growth with acidity signifies a positive presumptive reaction.

c. Interpretation: Production of an acidic reaction or gas in the tubes or bottles within 48 ± 3 h constitutes a positive presumptive reaction. Submit tubes with a positive presumptive reaction to the confirmed phase ( 9221B. 2).

9221: I. Preparation of Lauryl Tryptose Broth Inoculum mL Amount of Medium in Tube mL Volume of Medium + Inoculum mL Dehydrated Lauryl Tryptose Broth Required g/ L 1 10 or more 11 or more 35.6 10 10 20 71.2 10 20 30 53.4 20 10 30 106.8 100 50 150 106.8 100 35 135 137.1 100 20 120 213.6 The absence of acidic reaction or gas formation at the end of 48 ± 3 h of incubation constitutes a negative test. Submit drinking water samples demonstrating growth without a positive gas or acid reaction to the confirmed phase ( 9221B. 2). An arbitrary 48 h limit for observation doubtless excludes occasional members of the coliform group that grow very slowly ( see Section 9212).

2. Confirmed Phase a. Culture medium: Use brilliant green lactose bile broth fermentation tubes for the confirmed phase.

Brilliant green lactose bile broth:

Peptone ................................................................................... 10.0 g Lactose ................................................................................... 10.0 g Oxgall ..................................................................................... 20.0 g Brilliant green .................................................................... 0.0133 g Reagent grade water ................................................................ 1.0 L Add dehydrated ingredients to water, mix thoroughly, and heat to dissolve. pH should be 7.2 ± 0.2 after sterilization. Before sterilization, dispense, in fermentation tubes with an inverted vial, sufficient medium to cover inverted vial at least one half to two thirds after sterilization. Close tubes with metal or heat resistant plastic caps. Draft 3 June 2003 b. Procedure: Submit all presumptive tubes or bottles showing growth, any amount of gas, or acidic reaction within 24 ± 2 h of incubation to the confirmed phase. If active fermentation or acidic reaction appears in the presumptive tube earlier than 24 ± 2 h, transfer to the confirmatory medium; preferably examine tubes at 18 ± 1 h. If additional presumptive tubes or bottles show active fermentation or acidic reaction at the end of a 48 ± 3 h incubation period, submit these to the confirmed phase.

Gently shake or rotate presumptive tubes or bottles showing gas or acidic growth to resuspend the organisms. With a sterile loop 3.0 to 3.5 mm in diameter, transfer one or more loopfuls of culture to a fermentation tube containing brilliant green lactose bile broth or insert a sterile wooden applicator at least 2.5 cm into the culture, promptly remove, and plunge applicator to bottom of fermentation tube containing brilliant green lactose bile broth. Remove and discard applicator. Repeat for all other positive presumptive tubes.

Incubate the inoculated brilliant green lactose bile broth tube at 35 ± 0.5/ C. Formation of gas in any amount in the inverted vial of the brilliant green lactose bile broth fermentation tube at any time ( e. g., 6 ± 1 h, 24 ± 2 h) within 48 ± 3 h constitutes a positive confirmed phase. Calculate the MPN value from the number of positive brilliant green lactose bile tubes as described in Section 9221C.

c. Alternative procedure: Use this alternative only for polluted water or wastewater known to produce positive results consistently.

If all presumptive tubes are positive in two or more consecutive dilutions within 24 h, submit to the confirmed phase only the tubes of the highest dilution ( smallest sample inoculum) in which all tubes are positive and any positive tubes in still higher dilutions. Submit to the confirmed phase all tubes in which gas or acidic growth is produced only after 48 h.

3. Completed Phase To establish the presence of coliform bacteria and to provide quality control data, use the completed test on at least 10% of positive confirmed tubes ( see Figure 9221: 1). Simultaneous inoculation into brilliant green lactose bile broth for total coliforms and EC broth for fecal coliforms ( see Section 9221E below) or EC MUG broth for Escherichia coli may be used. Consider positive EC and EC MUG broths elevated temperature ( 44.5/ C) results as a positive completed test response. Parallel positive brilliant green lactose bile broth cultures with negative EC or EC MUG broth cultures indicate the presence of nonfecal coliforms. Draft 4 June 2003 a. Culture media and reagents:

1) LES Endo agar: See Section 9222B. Use 100 x 15 mm petri plates.

2) MacConkey agar:

Peptone ................................................................................ 17.0 g Proteose peptone .............................................................. .... 3.0 g Lactose ................................................................................ 10.0 g Bile salts .............................................................................. .1.5 g Sodium chloride. NaC1 ...................................................... .. 5.0 g Agar .................................................................................. .. 13.5 g Neutral red ........................................................................... 0.03 g Crystal violet ...................................................................... 0.001 g Reagent grade water ............................................................... 1.0 L Add ingredients to water, mix thoroughly, and heat to boiling to dissolve. Sterilize by autoclaving for 15 min at 121/ C. Temper agar after sterilization and pour into petri plates ( 100 X 15 mm). pH should be 7.1 ± 0.2 after sterilization.

3) Nutrient agar:

Peptone ................................................................................ .. 5.0 g Beef extract ............................................................................ 3.0 g Agar ...................................................................................... 15.0 g Reagent grade water ............................................................... 1.0 L Add ingredients to water, mix thoroughly, and heat to dissolve. pH should be 6.8 ± 0.2 after sterilization. Before sterilization. dispense in screw capped tubes. After sterilization, immediately place tubes in an inclined position so that the agar will solidify with a sloped surface. Tighten screw caps after cooling and store in a protected, cool storage area.

4) Gram stain reagents:

a) Ammonium oxalate crystal violet ( Hucker * s): Dissolve 2 g crystal violet ( 90% dye content) in 20 mL 95% ethyl alcohol; dissolve 0.8 g ( NH4) 2C2O4H2O in 80 mL reagentgrade water; mix the two solutions and age for 24 h before use; filter through paper into a staining bottle.

b) Lugol * s solution, Gram's modification: Grind 1 g iodine crystals and 2 g K1 in a mortar. Add reagent grade water, a few milliliters at a time, and grind thoroughly after each addition until solution is complete. Rinse solution into an amber glass bottle with the remaining water ( using a total of 300 mL).

c) Counterstain: Dissolve 2.5 g safranin dye in 100 mL 95% ethyl alcohol. Add 10 mL to 100 mL reagent grade water.

d) Acetone alcohol: Mix equal volumes of ethyl alcohol ( 95%) with acetone. Draft 5 June 2003 b. Procedure:

1) Using aseptic technique, streak one LES Endo agar ( Section 9222B. 2) or MacConkey agar plate from each tube of brilliant green lactose bile broth showing gas, as soon as possible after the observation of gas. Streak plates in a manner to insure presence of some discrete colonies separated by at least 0.5 cm. Observe the following precautions when streaking plates to obtain a high proportion of successful isolations if coliform organisms are present: ( a) Use a sterile 3 mm diam loop or an inoculating needle slightly curved at the tip; ( b) tap and incline the fermentation tube to avoid picking up any membrane or scum on the needle; ( c) insert end of loop or needle into the liquid in the tube to a depth of approximately 0.5 cm; and ( d) streak plate for isolation with curved section of the needle in contact with the agar to avoid a scratched or torn surface. Flame loop between second and third quadrants to improve colony isolation. Incubate plates ( inverted) at 35 ± 0.5/ C for 24 ± 2 h.

2) The colonies developing on LES Endo agar are defined as typical ( pink to dark red with a green metallic surface sheen) or atypical ( pink, red, white, or colorless colonies without sheen) after 24 h incubation. Typical lactose fermenting colonies developing on MacConkey agar are red and may be surrounded by an opaque zone of precipitated bile. From each plate pick one or more typical, well isolated coliform colonies or, if no typical colonies are present, pick two or more colonies considered most likely to consist of organisms of the coliform group, and transfer growth from each isolate to a single strength lauryl tryptose broth fermentation tube and onto a nutrient agar slant. ( The latter is unnecessary for drinking water samples.)

If needed, use a colony magnifying device to provide optimum magnification when colonies are picked from the LES Endo or MacConkey agar plates. When transferring colonies, choose well isolated ones and barely touch the surface of the colony with a flame sterilized, aircooled transfer needle to minimize the danger of transferring a mixed culture.

Incubate secondary broth tubes ( lauryl tryptose broth with inverted fermentation vials inserted) at 35 ± 0.5/ C for 24 ± 2 h; if gas is not produced within 24 ± 2 h reincubate and examine again at 48 ± 3 h. Microscopically examine Gram stained preparations from those 24 h nutrient agar slant cultures corresponding to the secondary tubes that show gas.

3) Gram stain technique   The Gram stain may be omitted from the completed test for potable water samples only because the occurrences of gram positive bacteria and spore forming organisms surviving this selective screening procedure are infrequent in drinking water.

Various modifications of the Gram stain technique exist. Use the following modification by Hucker for staining smears of pure culture: include a gram positive and a gram negative culture as controls.

Prepare separate light emulsions of the test bacterial growth and positive and negative control cultures on the same slide using drops of distilled water on the slide. Air dry and fix by passing slide through a flame and stain for 1 min with ammonium oxalate crystal violet solution. Rinse slide in tap water and drain off excess; apply Lugol* s solution for 1 min. Draft 6 June 2003 Rinse stained slide in tap water. Decolorize for approximately 15 to 30 s with acetone alcohol by holding slide between the fingers and letting acetone alcohol flow across the stained smear until the solvent flows colorlessly from the slide. Do not overdecolorize. Counterstain with safranin for 15 s, rinse with tap water, blot dry with absorbent paper or air dry, and examine microscopically. Gram positive organisms are blue; gram negative organisms are red. Results are acceptable only when controls have given proper reactions.

c. Interpretation: Formation of gas in the secondary tube of lauryl tryptose broth within 48 ± 3 h and demonstration of gram negative, nonspore forming, rod shaped bacteria from the agar culture constitute a positive result for the completed test, demonstrating the presence of a member of the coliform group. Draft 7 June 2003 4. Bibliography Meyer, E. M. 1918. An aerobic spore forming bacillus giving gas in lactose broth isolated in routine water examination. J. Bacteriol. 3: 9.

Hucker, G. J. & H. J. Conn. 1923. Methods of Gram Staining. N. Y. State Agr. Exp. Sm. Tech. Bull. No. 93.

Norton, J. F. & J. J. Weight. 1924. Aerobic spore forming lactose fermenting organisms and their significance in water analysis. Amer. J. Pub. Health 14: 1019.

Hucker, G. J. & H. J. Conn. 1927. Further Studies on the Methods of Gram Staining. N. Y. State Agr. Exp. Sta. Tech. Bull. No. 128.

Porter, R, C. S. McCleskey & M. Levine. 1937. The facultative sporulating bacteria producing gas from lactose. J. Bacteriol. 33: 163.

Cowles, P. B. 1939. A modified fermentation tube. J. Bacteriol. 38: 677.

Sherman, V. B. D. 1967. A Guide to the Identification of the Genera of Bacteria. Williams & Wilkins. Baltimore, Md.

Geldreich, E. E. 1975. Handbook for Evaluating Water Bacteriological Laboratories. 2nd ed. EPA 670/ 9 75 006. U. S. Environmental Protection Agency, Cincinnati, Ohio.

Evans, T. M.., C. E. Waarvick, R. J. Seidler & M. W. LeChevallier, 1981. Failure of the most probable number technique to detect coliforms in drinking water and raw water supplies. Appl. Environ. Microbiol. 41: 130.

Seidler, R. J., T. M. Evans, J. R. Kaufman, C. E. Waarvick & M. W. LeChevallier. 1981. Limitations of standard coliform enumeration techniques. J. Amer. Water Works Assoc. 73: 538.

Gerhards, P., ed. 1981. Manual of Methods for General Bacteriology. American Soc. Microbiology. Washington, D. C.

Krieg, N. R. & J. G. Holt, eds. 1984. Bergey's Manual of Systematic Bacteriology, Vol 1. Williams & Wilkins. Baltimore, Md.

Greenberg, A. E. & D. A. Hunt, eds. 1985. Laboratory Procedures for the Examination of Seawater and Shellfish. 5th ed. American Public Health Assoc. Washington, D. C.

U. S. Environmental Protection Agency. 1989. National primary drinking water regulations; analytical techniques; coliform bacteria; final rule. Federal Register 54( 135): 29998 ( July 17, 1989). Draft 8 June 2003 STANDARD METHODS 9221 C. ESTIMATION OF BACTERIAL DENSITY Reprinted by Permission from Supplement to the 20th Edition 1. Precision of Fermentation Tube Test Unless a large number of sample portions is examined, the precision of the fermentation tube test is rather low. For example, Table 9221: IV shows that the 95% confidence limits are often one third times and three times the estimate. Consequently, use caution when interpreting the sanitary significance of any single coliform result. When several samples from a given sampling point are estimated separately and the results combined in their geometric mean, the precision is greatly improved 2. Table Reading and Recording of MPN Record coliform concentration as the Most Probable Number ( MPN)/ 100 mL. The MPN values, for a variety of positive and negative combinations, are given in Tables 9221: II, III, and IV. The sample volumes indicated in Tables 9221: II and III are chosen especially for examining finished waters. Table 9221: IV illustrates MPN values for combinations of positive and negative results when five 10 mL, five 1.0 mL, and five 0.1 mL sample portion volumes are tested. If the sample portion volumes used are those found in the tables, report the value corresponding to the number of positive and negative results in the series as the MPN/ 100 mL, or, when appropriate, as presence or absence. When the series of decimal dilutions is different from that in the table, select the MPN value from Table 9221: IV for the combination of positive results and calculate according to the following formulas:

MPN/ l00 mL = MPN value ( from table) × 10/ V where:

V = volume of one sample portion at the lowest selected dilution.

When more than three dilutions are used in a decimal series of dilutions, select the three most appropriate dilutions and refer to Table 9221: IV. Several examples are shown in the following table.

Volume mL Example 10 mL 1 mL 0.1 mL 0.01 mL 0.001 mL Combination of positives MPN Index No./ 100 mL a 5 5 1 0 0 5 1 0 330 b 4 5 1 0 0 4 5 1 48 c 0 0 1 0 0 0 0 1 1.8 d 5 4 4 1 0 4 4 1 400 e 5 4 4 0 1 4 4 1 400 f 5 5 5 5 2 5 5 2 54,000 Draft 9 June 2003 Select highest dilution that gives positive results in all five portions tested ( no lower dilution giving any negative results) and the two next succeeding higher dilutions ( Example a). If the lowest dilution tested has less than five portions with positive results, select it and the two next succeeding higher dilutions ( Examples b, c).

When a positive result occurs in a dilution higher than the three selected according to the fore going rules, change the selection to the lowest dilution that has less than five positive results and the next two higher dilutions ( Example d). When all the foregoing selection rules have left unselected any higher dilutions with positive results, add those higher dilution positive results to the tested to select three dilutions, then select the next lower dilution ( Example f).

When it is desired to summarize with a single MPN value the results from several samples, use the geometric mean or the median. The geometric mean is computed by averaging the logarithmic values; e. g., the geometric mean of A, B, and C is 10L where L = ( log10A + log10B + log10C)/ 3 Mean values are reported as the antilog of L.

Table 9221: IV shows all but the very improbable positive tube combinations for a three dilution series. In testing 10 different samples, there is a 99% chance of finding all the results among these 95 outcomes. If untabulated combinations occur with a frequency greater than 0.1%, it indicates that the technique is faulty or that the statistical assumptions underlying the MPN estimate are not being fulfilled ( e. g., growth inhibition at low dilutions).

The MPN for combinations not appearing in the table, or for other combinations of tubes or dilutions, may be estimated as follows: First select the lowest dilution that does not have all positive results. Second, select the highest dilution with at least one positive result. Finally, select all the dilutions between them. For example, from ( 5/ 5, 10/ 10, 4/ 10, 1/ 10, 0/ 5) use only (, , 4/ 10, 1/ 10, ), 4/ 10 @ 0.1 mL sample/ tube and 1/ 10 @ 0.01 mL sample/ tube; from ( 5/ 5, 10/ 10, 10/ 10, 0/ 10, 0/ 5) select only (,, 10/ 10, 0/ 10, ), 10/ 10 @ 0.1 mL sample/ tube and 0/ 10 @ 0.01 mL sample/ tube. Use only the selected dilutions in the following formula of Thomas1 :

MPN/ 100 mL ( approx.) = 100 × P/( N × T) 1/ 2 where:

P = number of positive results, N = volume of sample in all the negative portions combined, mL, and T = total volume of sample in the selected dilutions, mL.

In the first example above,

MPN/ 100 mL ( approx.) = 100 × 5/ ( 0.69 × 1.1) 1/ 2 = 500/ 0.87 = 570/ 100 mL In the second example above,

MPN/ 100 mL ( approx) = 100 × 10/( 0.1 × 1.1) 1/ 2 = 1000/ 0.332 = 3000/ 100 mL Draft 10 June 2003 The two examples compare well with the true MPNs, 590/ 100 mL and 2400/ 100 mL, respectively. The second example is a special case for which an exact solution can be calculated directly for the two selected dilutions.

Although MPN tables and calculations are described for use in the coliform test, they also determine the MPN of any other organisms provided that suitable test media are available.

When all the results at the lower dilutions are positive and all the results at higher dilutions are negative, it is possible to calculate an exact MPN for two selected dilutions as follows: When V is the volume of each individual sample portion at the highest dilution with all positive portions,

MPN/ 100 mL = ( 1/ V) [ 230.3 log10 ( T/ N)]

where T and N are defined as for Thomas's formula. The last example discussed above was ( 5/ 5, 10/ 10, 10/ 10, 0/ 10, 0/ 5), with portions 10, 1, 0.1, 0.01, and 0.001. The third dilution is the highest with positive portions, so V = 0.1. The MPN for the third and fourth dilution would be exactly MPN/ 100 mL = ( 1/ 0.1) × [ 230.3 log10 ( 1.1/ 0.1)]

= 2400/ 100 mL 3. Reference Thomas, H. A., JR. 1942. Bacterial densities from fermentation tube tests. J. Amer. Water Works Assoc. 34: 572.

4. Bibliography McCrady, M. H. 1915. The numerical interpretation of fermentation tube results. J. Infect. Dis. 12: 183.

McCrady, M. H. 1918. Tables for rapid interpretation of fermentation tube results. Pub. Health J. 9: 201.

Hoskins, J. K. 1933. The most probable numbers of B. coli in water analysis. J. Amer. Water Works Assoc. 25: 867.

Hoskins, J. K. 1934. Most Probable Numbers for evaluation of coli aerogenes tests by fermentation tube method. Pub. Health Rep. 49: 393.

Halvorson, H. O. & N. R. Ziegler. 1933 35. Application of statistics to problems in bacteriology. J. Bacteriol. 25: 101: 26: 331.559; 29: 609.

Eisenhart, C. & P. W. Wilson. 1943. Statistical methods and control in bacteriology. Bacteriol. Rev. 7: 57.

Cochran, W. G. 1950. Estimationof bacterial densities by means of the " Most Probable Number." Biometrics 6: 105.

Woodward, R. L. 1957. How probable is the Most Probable Number? J. Amer. Water Works Assoc. 49: 1060.

DeMan, J. C. 1983. MPN tables, corrected. Eur. J. Appl. Biotechnol. 17: 301.

Garthright, W. E. 1998. Appendix 2. Most probable number form serial dilutions. FDA Bacteriological Analytical Manual, 8th ed., Rev. A. AOAC International, Gaithersburg, Md. Draft 11 June 2003 Table 9221: II. MPN Index and 95% Confidence Limits for Various Combinations of Positive and Negative Results When Five 20 mL Portions Are Used No. of Tubes Giving Positive Reaction Out of 5 ( 20mLEach) MPN Index/ I00 mL 95% Confidence Limits ( Exact)

Lower Upper 0 < 1.1   3.5 I 1.1 0.051 5.4 2 2.6 0.4 8.4 3 4.6 1.0 13 4 8.0 2.1 23 5 > 8.0 3.4   Table 9221. III. MPN Index and 95% Confidence Limits for Various Combinations of Positive and Negative Results When Ten 10 mL Portions Are Used No. of Tubes Giving Positive Reaction Out of 10 ( 10 mL Each) MPN Index/ 100 mL 95% Confidence Limits ( Exact)

Lower Upper 0 < 1.1   3.4 1 1.1 0.051 5.9 2 2.2 0.37 8.2 3 3.6 0.91 9.7 4 5.1 1.6 13 5 6.9 2.5 15 6 9.2 3.3 19 7 12 4.8 24 8 16 5.8 34 9 23 8.1 53 10 > 23 13   Draft 12 June 2003 Table 9221. IV. MPN Index and 95% Confidence Limits for Various Combinations of Positive Results When Five Tubes Are Used per Dilution ( 10 mL, 1.0 mL, 0.1 mL)*

Combination of Positives MPN Index/ 100 mL Confidence Limits Combination of Positives MPN Index/ 100 mL Confidence Limits Low High Low High 0 0 0 0 0 1 0 1 0 0 1 1 0 2 0 0 2 1 0 3 0 1 0 0 1 0 1 1 0 2 1 1 0 1 1 1 1 1 2 1 2 0 1 2 1 1 3 0 1 3 1 1 4 0 2 0 0 2 0 1 2 0 2 2 1 0 2 1 1 2 1 2 2 2 0 2 2 1 2 2 2 2 3 0 2 3 1 2 4 0 3 0 0 3 0 1 3 0 2 3 1 0 3 1 1 3 1 2 3 2 0 3 2 1 3 2 2 3 3 0 3 3 1 3 3 2 3 4 0 3 4 1 3 5 0 4 0 0 4 0 1 4 0 2 < 1.8 1.8 1.8 3.6 3.7 5.5 5.6 2.0 4.0 6.0 4.0 6.1 8.1 6.1 8.2 8.3 10 10 4.5 6.8 9.1 6.8 9.2 12 9.3 12 14 12 14 15 7.8 11 13 11 14 17 14 17 20 17 21 24 21 24 25 13 17 21   0.090 0.090 0.70 0.70 1.8 1.8 0.10 0.70 1.8 0.71 1.8 3.4 1.8 3.4 3.4 3.5 3.5 0.79 1.8 3.4 1.8 3.4 4.1 3.4 4.1 5.9 4.1 5.9 5.9 2.1 3.5 5.6 3.5 5.6 6.0 5.7 6.8 6.8 6.8 6.8 9.8 6.8 9.8 9.8 4.1 5.9 6.8 6.8 6.8 6.9 10 10 15 15 10 10 15 12 15 22 15 22 22 22 22 15 15 22 17 22 26 22 26 36 26 36 36 22 23 35 26 36 36 36 40 40 40 40 70 40 70 70 35 36 40 4 0 3 4 1 0 4 1 1 4 1 2 4 1 3 4 2 0 4 2 1 4 2 2 4 2 3 4 3 0 4 3 1 4 3 2 4 4 0 4 4 1 4 4 2 4 5 0 4 5 1 5 0 0 5 0 1 5 0 2 5 0 3 5 1 0 5 1 1 5 1 2 5 1 3 5 2 0 5 2 1 5 2 2 5 2 3 5 2 4 5 3 0 5 3 1 5 3 2 5 3 3 5 3 4 5 4 0 5 4 1 5 4 2 5 4 3 5 4 4 5 4 5 5 5 0 5 5 1 5 5 2 5 5 3 5 5 4 5 5 5 25 17 21 26 31 22 26 32 38 27 33 39 34 40 47 41 48 23 31 43 58 33 46 63 84 49 70 94 120 150 79 110 140 170 210 130 170 220 280 350 430 240 350 540 920 1600 > 1600 9.8 6.0 6.8 9.8 10 6.8 9.8 10 14 9.9 10 14 14 14 15 14 15 6.8 10 14 22 10 14 22 34 15 22 34 36 58 22 34 52 70 70 36 58 70 100 100 150 70 100 150 220 400 700 70 40 42 70 70 50 70 70 100 70 70 100 100 100 120 100 120 70 70 100 150 100 120 150 220 150 170 230 250 400 220 250 400 400 400 400 400 440 710 710 1100 710 1100 1700 2600 4600   * Results to two significant figures Draft 13 June 2003 STANDARD METHODS 9221 E. FECAL COLIFORM PROCEDURE Reprinted by Permission from the 20th Edition Elevated temperature tests for distinguishing organisms of the total coliform group that also belong to the fecal coliform group are described herein. Modifications in technical procedures. standardization of methods, and detailed studies of the fecal coliform group have established the value of this procedure. The test can be performed by one of the multiple tube procedures described here or by membrane filter methods as described in Section 9222. The procedure using A l broth is a single step method.

The fecal coliform test ( using EC medium) is applicable to investigations of drinking water, stream pollution, raw water sources, wastewater treatment systems, bathing waters, seawaters, and general waterquality monitoring. Prior enrichment in presumptive media is required for optimum recovery of fecal coliforms when using EC medium. The test using A 1 medium is applicable to source water, seawater, and treated wastewater.

1. Fecal Coliform Test ( EC Medium)

The fecal coliform test is used to distinguish those total coliform organisms that are fecal coliforms. Use EC medium or, for a more rapid test of the quality of shellfish waters, treated wastewaters, or source waters, use A 1 medium in a direct test.

a. EC medium:

Tryptose or trypticase .............................................................. 20.0 g Lactose ....................................................................................... 5.0 g Bile salts mixture or bile salts No. .......................................... 3 1.5 g Dipotassium hydrogen phosphate. K2HPO4 ............................... 4.0 g Potassium dihydrogen phosphate. KH2PO4 ................................ 1.5 g Sodium chloride. NaCl ............................................................... 5.0 g Reagent grade water ................................................................... 1.0 L Add dehydrated ingredients to water, mix thoroughly, and heat to dissolve. pH should be 6.9 ± 0.2 after sterilization. Before sterilization, dispense in fermentation tubes, each with an inverted vial, sufficient medium to cover the inverted vial at least partially after sterilization. Close tubes with metal or heat resistant plastic caps.

b. Procedure: Submit all presumptive fermentation tubes or bottles showing any amount of gas, growth, or acidity within 48 h of incubation to the fecal coliform test.

1) Gently shake or rotate presumptive fermentation tubes or bottles showing gas, growth, or acidity. Using a sterile 3 or 3.5 mm diam loop or sterile wooden applicator stick, transfer growth from each presumptive fermentation tube or bottle to EC broth ( see Section 9221B. 2).

2) Incubate inoculated EC broth tubes in a water bath at 44.5 ± 0.2/ C for 24 ± 2 h.

Place all EC tubes in water bath within 30 min after inoculation. Maintain a sufficient water depth in water bath incubator to immerse tubes to upper level of the medium. Draft 14 June 2003 c. Interpretation: Gas production with growth in an EC broth culture within 24 ± 2 h or less is considered a positive fecal coliform reaction. Failure to produce gas ( with little or no growth) constitutes a negative reaction. If multiple tubes are used, calculate MPN from the number of positive EC broth tubes as described in Section 9221C. When using only one tube for subculturing from a single presumptive bottle, report as presence or absence of fecal coliforms.

2. Fecal Coliform Direct Test ( A 1 Medium)

a. A 1 broth: This medium may be used for the direct isolation of fecal coliforms from water. Prior enrichment in a presumptive medium is not required.

Lactose ..................................................................................... 5.0 g Tryptone.................................................................................. 20.0 g Sodium chloride. NaCl ............................................................. 5.0 g Salicin ....................................................................................... 0.5 g Polyethylene glycol p isooctylphenyl ether* ......................... 1.0 mL Reagent grade water .................................................................. 1.0 L * Triton X l00. Rohm and Haas Co., or equivalent.

Heat to dissolve solid ingredients, add polyethylene glycol p isooctyiphenyl ether, and adjust to pH 6.9 ± 0.1. Before sterilization dispense in fermentation tubes with an inverted vial sufficient medium to cover the inverted vial at least partially after sterilization. Close with metal or heat resistant plastic caps. Sterilize by autoclaving at 121/ C for 10 min. Store in dark at room temperature for not longer than 7 d. Ignore formation of precipitate.

Make A 1 broth of such strength that adding l0 mL sample portions to medium will not reduce ingredient concentrations below those of the standard medium. For l0 mL samples prepare double strength medium.

b. Procedure: Inoculate tubes of A 1 broth as directed in Section 9221B. 1b1). Incubate for 3 h at 35 ± 0.5/ C. Transfer tubes to a water bath at 44.5 ± 0.2/ C and incubate for an additional 21 ± 2 h.

c. Interpretation: Gas production in any A 1 broth culture within 24 h or less is a positive reaction indicating the presence of fecal coliforrns. Calculate MPN from the number of positive A 1 broth tubes as described in Section 9221C. Draft 15 June 2003 3. Bibliography Perry, C. A. & A. A. Haina. 1933. A modified Eijkman medium. J. Bacteriol. 26: 419.

Perry, C. A. & A. A. Haina. 1944. Further evaluation of EC medium for the isolation of coliform bacteria and Eschenchia coli. Amer. J. Pub. Health 34: 735.

Geldreich, E. E., H. F. Clark, P. W., Kabler, C. B. Huff & R. H. Bordner. 1958. The coliform group. II. Reactions in EC medium at 45/ C. Appl. Microbiol. 6: 347.

Geldreich, E. E., R. H. Bordner, C. B. Huff, H. F. Clark & P. W. Kabler. 1962. Type distribution of coliform bacteria in the feces of warmblooded animals. J. Water Pollut. Control Fed. 34: 295.

Geldreich, E. E. 1966. Sanitary significance of fecal coliforms in the environment. FWPCA Publ. WP 20 3 ( Nov.). U. S. Dep. Interior, Washington, D. C.

Andrews, W. H. & M. W. Presnell. 1972. Rapid recovery of Eschenchia coli from estuarine water. Appl. Microbiol. 23: 521.

Olson, B. H. 1978. Enhanced accuracy of coliform testing in seawater by a modification of the mostprobable number method. Appl. Microbiol. 36: 438.

Strandridge, J. H. & J. J. Delfino. 1981. A 1 Medium: Alternative technique for fecal coliform organism enumeration in chlorinated wastewaters. Appl. Environ. Microbiol. 42: 9 18. Draft 16 June 2003 STANDARD METHODS 9221 F. ESCHERICHIA COLI PROCEDURE Reprinted by Permission from the Supplement to the 20th Edition  Proposed Escherichia coli is a member of the fecal coliform group of bacteria. This organism in water indicates fecal contamination. Enzymatic assays have been developed that allow for the identification of this organism. Assays for $ glucuronidase or glutamate decarboxylase may be used to determine the presence of E. coli. In method 9221F. 1, E. coli are defined as coliform bacteria that possess the enzyme $ glucuronidase and are capable of cleaving the fluorogenic substrate 4 methylumbel liferyl$ Dglucuronide ( MUG) with the corresponding release of the fluorogen when grown in EC MUG medium at 44.5/ C within 24 ± 2 h or less. In method 9221F. 2, E. coli are defined as coliform bacteria that possess the enzyme glutamate decarboxylase and are capable of producing an alkaline reaction within 4 h in a reagent containing a lytic agent and glutamic acid. The procedure is used as a confirmatory test after prior enrichment in a presumptive medium for total coliform bacteria. These tests are performed by the tube procedure described here or by the membrane filter method as described in Section 9222. The chromogenic substrate procedure ( Section 9223) can be used for direct detection of E. coli.

Tests for E. coli are applicable for the analysis of drinking water, surface and ground water, and wastewater. E. coli is a member of the indigenous fecal flora of warm blooded animals. The occurrence of E. coli is considered a specific indicator of fecal contamination and the possible presence of enteric pathogens.

1. Escherichia coli Test ( EC MUG medium)

Use EC MUG medium for the confirmation of E. coli.

a. EC MUG medium:

Tryptose or trypticase ............................................................... .20.0 g Lactose ........................................................................................ .5.0 g Bile salts mixture or bile salts No. .......................................... .. 31.5 g Dipotassium hydrogen phosphate. K2HPO4 .............................. .. 4.0 g Potassium dihydrogen phosphate, KH2PO4 ................................. 1.5 g Sodium chloride, NaC1 ............................................................. .. 5.0 g 4 methylumbelliferyl$ D glucuronide ( MUG) ......................... 0.05 g Reagent grade water ..................................................................... 1.0 L Add dehydrated ingredients to water, mix thoroughly, and heat to dissolve. pH should be 6.9 ± 0.2 after sterilization. Before sterilization, dispense in tubes that do not fluoresce under long wavelength ( 366 nm) ultraviolet ( UV) light. An inverted tube is not necessary. Close tubes with metal or heat resistant plastic caps.

b. Procedure: Submit all presumptive fermentation tubes or bottles showing growth, gas, or acidity within 48 ± 3 h of incubation to the E. coli test.

1) Gently shake or rotate presumptive fermentation tubes or bottles showing growth. gas, or acidity. Using a sterile 3 or 3.5 mm diam metal loop or sterile wooden applicator stick, transfer growth from presumptive fermentation tube or bottle to EC MUG broth. Draft 17 June 2003 2) Incubate inoculated EC MUG tubes in a water bath or incubator maintained at 44.5 ± 0.2/ C for 24 ± 2 h. Place all EC MUG tubes in water bath within 30 min after inoculation. Maintain a sufficient water depth in the water bath incubator to immerse tubes to upper level of medium.

c. Interpretation: Examine all tubes exhibiting growth for fluorescence using a longwavelength UV lamp ( preferably 6 W). The presence of bright blue fluorescence is considered a positive response for E. coli. A positive control consisting of a known E. coli ( MUG positive) culture, a negative control consisting of a thermotolerant Klebsiella pneumoniae ( MUG negative) culture, and an uninoculated medium control may be necessary to interpret the results and to avoid confusion of weak auto fluorescence of the medium as a positive response. If multiple tubes are used, calculate MPN from the number of positive ECMUG broth tubes as described in Section 9221C. When using only one tube or subculturing from a single presumptive bottle, report as presence or absence of E. coli.

2. Escherichia coli Test ( GAD Procedure)

The GAD test procedure is not approved for use in LT2 monitoring.

3. Bibliography Feng, P. C. S. & P. A. Hartman. 1982. Fluorogenic assays for immediate confirmation of Escherichia coli. Appl. Environ. Microbiol. 43: 1320.

Hartman, P. A. 1989. The MUG ( glucuronidase) test for E. coli in food and water. In A. Balows et al., eds., Rapid Methods and Automation in Microbiology and Immunology. Proc. 5th Intl. Symp. on Rapid Methods and Automation in Microbiology & Immunology, Florence, Italy, Nov. 4 6, 1987.

Fieldler, J. & J. Reiske. 1990. Glutaminsauredecarboxylase schnelltest zur identifikation von Escherichia coli. Z. Ges. Hyg. Grenzgeb. 36: 620.

Shadix, L. C. & E. W. Rice. 1991. Evaluation of $ glucuronidase assay for the detection of Escherichia coli from environmental waters. Can. J. Microbiol. 37: 908.

Rice, E. W., C. H. Johnson, M. E. Dunnigan & D. J. Reasoner. 1993. Rapid glutamate decarboxylase assay for the detection of Escherichia coli. Appl. Environ. Microbiol. 59: 4937. Errata. 1995. Appl. Environ. Microbiol. 61: 847.

Rice, E. W., C. H. Johnson, & D. J. Reasoner. 1996. Detection of Escherichia coli O157: H7 in water from coliform enrichment cultures. Lett. Appl. Microbiol. 23: 179.


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$ 31.06/ hr Respon.

hr/ yr Labor Cost/ yr Capital Startup Cost O & M Cost( 1) Applications/

respondent ( 2) Number of Respon. Total hr/ yr Total Cost/ yr Review of regs and guidance document 20 9 10 4 0 1 44 3,459 0 10 1.0 74 3,256 256,728 Developing eng families groups 20 3 1 0 0 1 25 1,716 0 0 1.0 74 1,850 127,012 Gathering durability data 12 5 0 38 2 3 60 3,149 0 0 2.5 74 11,100 582,621 Testing/ Gathering emission data on test engines 30 14 0 22 2 14 82 4,794 0 0 2.5 74 15,170 886,934 Laboratory maintenance( 1) 0 0 0 0 0 0 0 0 0 6,200 1.0 34 0 210,800 Test Cost ( annualized)( 1) 0 0 0 0 0 0 0 0 0 917 1.7 27 0 42,090 Analyze data to determine compliance 11 6 1 0 0 1 19 1,429 0 0 2.5 74 3,515 264,317 Preparing and submitting certification application 14 1 0 0 0 4 19 1,073 0 4 2.5 74 3,423 199,178 Preparing and submitting " carry over" applications 1 0 0 0 0 2 3 126 0 2 6.6 74 1,465 62,711 Fee ( 3) 0 0 0 0 0 0 0 0 0 826 9.1 74 0 556,228 Preparing and submitting evaporative emissions information ( 4) 20 0 0 20 2 2 44 2,330 0 826 2.5 74 8,140 583,949 Preparing and supporting running changes 7 5 1 3 0 2 18 1,242 0 6 3.1 74 4,129 286,273 Store, file and maintain records 3 1 1 0 0 7 12 616 0 5 9.1 74 8,081 418,222 Total per respondent 138 44 14 87 6 37 326 19,935 0 varies varies N/ A N/ A N/ A Total for the industry N/ A N/ A N/ A N/ A N/ A N/ A N/ A $ 3,507,934 0 $ 969,129 varies 74 60,129 $ 4,477,063 ( 1) See section 6( b) ii for details. ( 3) See section 6( b)( ii) for details. Average Burden= total burden/# respond ( 2) See section 6( d) for details. ( 4) Large SI engines only. = 813 hrs per respon Table 1 Annual Respondent Burden and Cost  SI Engine Certification Hours and cost per application Small and Large SI Engines Total hours and cost Information Collection Activity Engineer @ 64.28/ hr Manager @

$ 96.96/ hr Legal @

$ 108.84/ hr Test Cell Operator@

$ 45.40/ hr Transportati on @

$ 37.38/ hr Clerical@

$ 31.06/ hr Respon.

hr/ yr Labor Cost/ yr Capital Startup Cost O & M Cost( 1) Applications/ r espondent ( 2) Number of Respon. Total hr/ yr Total Cost/ yr Review of regs and guidance document 20 9 10 4 0 1 44 3,459 0 10 1.0 32 1,408 111,018 Developing eng families groups 20 3 1 0 0 1 25 1,716 0 0 1.0 32 800 54,924 Gathering durability data 12 5 0 38 2 3 60 3,149 0 0 3.1 32 5,952 312,411 Testing/ Gathering emission data on test engines 30 14 0 22 2 14 82 4,794 0 0 15.0 8 9,840 575,309 Laboratory maintenance( 1) 0 0 0 0 0 0 0 0 0 6,224 1.0 8 0 49,792 Test Cost ( annualized)( 1) 0 0 0 0 0 0 0 0 0 500 1.3 23 0 14,950 Analyze data to determine compliance 11 6 1 0 0 1 19 1,429 0 0 3.1 32 1,885 141,731 Preparing and submitting certification application 14 1 0 0 0 4 19 1,073 0 4 1.4 32 829 48,233 Preparing and submitting " carry over" applications 1 0 0 0 0 2 3 126 0 2 1.6 32 154 6,574 Fee ( 3) 0 0 0 0 0 0 0 0 0 826 4.7 32 0 124,230 Preparing and supporting running changes 7 5 1 3 0 2 18 1,242 0 6 3.1 10 558 38,686 Store, file and maintain records 3 1 1 0 0 7 12 616 0 5 4.7 32 1,805 93,407 Total per respondent 118 44 14 67 4 35 282 17,605 0 varies varies N/ A N/ A N/ A Total for the industry N/ A N/ A N/ A N/ A N/ A N/ A N/ A $ 1,380,753 0 $ 190,512 varies 32 23,230 $ 1,571,265 ( 1) See section 6( b) ii for details. ( 3) See section 6( b)( ii) for details. Average Burden= total burden/# respond ( 2) See section 6( d) for details. = 726 hrs per respon Table 2 Annual Respondent Burden and Cost  SI Engine Certification Hours and cost per application Recreational Vehicles Information Collection Activity Engineer @ 64.28/ hr Manager @

$ 96.96/ hr Legal @

$ 108.84/ h r Clerical@

$ 31.06/ hr Respon.

hr/ yr Labor Cost/ yr Capital Startup Cost O & M Cost ( 1) Number of Respon. Total hr/ yr Total Cost/ yr Precertification activities/ Submit info in cert application 60 2 2 0 64 4,268 0 20 31 1,984 132,940 Gather Information regarding point of first retail sale ( 2) 160 3 1 24 188 11,430 0 50 31 5,828 355,879 Develop and submit end of year report 35 2 2 4 43 2,786 0 20 31 1,333 86,975 Develop and submit final reports 20 1 1 4 26 1,616 0 10 31 806 50,395 Recordkeeping ( 2) 24 4 28 1,978 0 10 31 868 61,630 Total 299 8 10 32 349 22,078 0 110 N/ A N/ A N/ A Total for the industry N/ A N/ A N/ A N/ A N/ A $ 684,409 0 $ 3,410 31 10,819 $ 687,819 ( 1) See section 6( b) ii for details.

( 2) Numbers reflect one activity done four times a year. Table 3 Annual Respondent Burden and Cost  Average, Trading and Banking Program Small SI Engines and Recreational Vehicles Hours and cost per application Total hours and cost Table 4 Annual Respondent Burden and Cost  Production Line Testing Program Small SI Phase 2 and Large SI Engines Information Collection Activity Engineer @ 64.28/ hr Manager @

$ 96.96/ hr Legal @

$ 108.84/ hr Clerical@

$ 31.06/ hr Test Cell Operator @

$ 45.40/ hr Assembler @

$ 33.60/ hr Transport @

$ 37.88/ hr Respon.

hr/ yr Labor Cost/ yr Capital Startup Cost O & M Cost( 1) Frequency ( 2) Number of Respon. Total hr/ yr Total Cost/ yr Review of instructions and regulations 45 5 8 0 0 0 0 58 4,248 0 0 1 37 2,146 157,180 Training 8 0 0 0 1 0 0 9 560 0 30 1 37 333 21,817 Projecting testing needs and planning test schedules 11 1 0 1 0 0 0 13 835 0 5 1 37 481 31,084 Engine selection and transport 4 0 0 0 1 1 1 7 374 0 1 1 37 259 13,857 Engine inspection 1 0 0 0 6 0 0 7 337 0 0 4 37 1,036 49,829 Testing ( In house) 8 0 0 0 12 2 0 22 1,126 0 100 10 36 7,920 441,446 Data entry and analysis 8 0 0 1 0 0 0 9 545 0 5 4 36 1,296 79,243 Other tasks ( test equipment calibration, engine repair, etc.) 4 0 0 0 60 0 0 64 2,981 0 5 1 36 2,304 107,500 Testing ( Contract Out) 0 0 0 0 0 0 0 0 0 0 2,750 30 1 0 82,500 Setting up contract 2 1 2 2 0 0 0 7 505 0 20 1 1 7 525 Preparing and submitting report 8 1 1 2 0 0 0 12 782 0 5 4 37 1,776 116,500 Store, file and maintain records 2 0 0 3 0 1 0 6 255 0 2 4 37 888 38,086 Total per manufacturer 101 8 11 9 80 4 1 214 12,549 0 varies varies N/ A N/ A N/ A Total for the industry N/ A N/ A N/ A N/ A N/ A N/ A N/ A N/ A $ 1,017,779 0 $ 121,788 N/ A 37 18,446 1,139,567 ( 1) Includes diskettes, photocopying, postage expenses, phone calls, and testing costs, annualized. See section 6( b)( iii) for details.

( 2) 1 = one time tasks; 4 = tasks carried out quarterly; other # = number of engine families. Refer to Section 6( d) for further detail. Average Burden= total burden/# respond = 499 hrs per respon Total hours and cost Hours and cost per engine family Table 5 Annual Respondent Burden and Cost  Production Line Testing Program Recreational Vehicles Information Collection Activity Engineer @ 64.28/ hr Manager @

$ 96.96/ hr Legal @

$ 108.84/ hr Clerical@

$ 31.06/ hr Test Cell Operator @

$ 45.40/ hr Assembler @

$ 33.60/ hr Transport @

$ 37.88/ hr Respon.

hr/ yr Labor Cost/ yr Capital Startup Cost O & M Cost( 1) Frequency ( 2) Number of Respon. Total hr/ yr Total Cost/ yr Review of instructions and regulations 45 5 8 0 0 0 0 58 4,248 0 0 1 32 1,856 135,940 Training 8 0 0 0 1 0 0 9 560 0 30 1 32 288 18,868 Projecting testing needs and planning test schedules 11 1 0 1 0 0 0 13 835 0 5 1 32 416 26,883 Engine selection and transport 4 0 0 0 1 1 1 7 374 0 1 1 32 224 11,984 Engine inspection 1 0 0 0 6 0 0 7 337 0 0 4 32 896 43,095 Testing ( In house) 8 0 0 0 12 2 0 22 1,126 0 100 14 8 2,464 137,339 Data entry and analysis 8 0 0 1 0 0 0 9 545 0 5 4 8 288 17,610 Other tasks ( test equipment calibration, engine repair, etc.) 4 0 0 0 60 0 0 64 2,981 0 5 1 8 512 23,889 Testing ( Contract Out) 0 0 0 0 0 0 0 0 0 0 2,750 4 23 0 246,675 Setting up contract 2 1 2 2 0 0 0 7 505 0 20 1 23 161 12,082 Preparing and submitting report 8 1 1 2 0 0 0 12 782 0 5 4 32 1,536 100,756 Store, file and maintain records 2 0 0 3 0 1 0 6 255 0 2 4 32 768 32,940 Total per manufacturer 101 8 11 9 80 4 1 214 12,549 0 varies varies N/ A N/ A N/ A Total for the industry N/ A N/ A N/ A N/ A N/ A N/ A N/ A N/ A $ 547,478 0 $ 260,583 N/ A 32 9,409 808,061 ( 1) Includes diskettes, photocopying, postage expenses, phone calls, and testing costs, annualized. See section 6( b)( iii) for details.

( 2) 1 = one time tasks; 4 = tasks carried out quarterly; other # = number of engine families. Refer to Section 6( d) for further detail. Average Burden= total burden/# respond = 294 hrs per respon Hours and cost per engine family Total hours and cost Information Collection Activity Engineer @ 64.28/ hr Manager @

$ 96.96/ hr Legal @

$ 108.84/ hr Clerical@

$ 31.06/ hr Test Cell Operator@

$ 45.40/ hr Assembler @

$ 33.60/ hr Transport @

$ 37.88/ hr Respon.

hr/ yr Labor Cost/ yr Capital Startup Cost O & M Cost( 1) Frequency ( 2) Number of Respon. Total hr/ yr Total Cost/ yr Review of instructions and regulations 7 1 1 0 0 0 0 9 656 0 0 1 32 288 20,984 Training 2 0 0 0 1 0 0 3 174 0 30 1 32 96 6,527 Plan activities 16 0 0 0 0 0 0 16 1,028 0 30 1 32 512 33,871 Procure engines 2 1 0 2 0 0 0 5 288 0 20 1 32 160 9,844 Ship Engines 1 0 0 0 0 0 4 5 214 0 100 1 32 160 10,042 Engine Maintenance 1 0 0 0 3 33 0 37 1,309 0 25 1 32 1,184 42,697 Testing ( Production < 75K engines) In house 8 0 0 0 38 1 1 48 2,310 0 100 2 17 1,632 81,954 Testing ( Production < 75K engines) Contract Out 0 0 0 0 0 0 0 0 0 0 2,750 2 9 0 49,500 Setting up contract 2 1 2 2 0 0 0 7 505 0 10 1 9 63 4,638 Testing ( Production 75k+

engines) 8 0 0 0 38 1 1 48 2,310 0 100 16 6 4,608 231,400 Data entry and analysis 22 0 0 5 2 0 0 29 1,660 0 1 1 32 928 53,160 Preparing and submitting report 18 1 1 1 0 0 0 21 1,394 0 3 1 32 672 44,701 Store, file and maintain records 8 0 0 1 0 0 0 9 545 0 1 1 32 288 17,482 Total per manufacturer 95 4 4 11 82 35 6 237 varies 0 varies varies N/ A N/ A N/ A Total for the industry N/ A N/ A N/ A N/ A N/ A N/ A N/ A N/ A $ 537,491 0 $ 69,310 N/ A 32 10,591 606,801 ( 1) Includes diskettes, photocopying, postage expenses, phone calls, and testing costs, annualized.

See section 6( b)( iii) for details.

( 2) 1 = one time tasks; 4 = tasks carried out quarterly; other # = number of tests performed/ eng manufacturer/ model year Refer to Section 6( d) for further detail.

average burden= total burden/# respond = 330.96875 hrs per respon Average Burden In use program( total burden Phase 1 + Phase 2)/(# respond Phase1 + Phase2)

= 353.73529 hrs per respon Annual Respondent Burden and Cost  Phase 1 In useTesting Program Table 6 Total hours and cost Hours and cost per application Information Collection Activity Technical @

$ 60.61/ hr Manager @

$ 91.01/ hr Legal @

$ 92.80 / hr Clerical @

29.86/ hr Test Cell @

$ 45.28/ hr Assembler @

$ 33.60/ hr Transport @

$ 38.62/ hr Respon.

hr/ yr Labor Cost/ yr Capital Startup Cost O & M Cost( 1) Frequency ( 2) Number of Respon. Total hr/ yr Total Cost/ yr Review of instructions and regulations 7 1 1 0 0 0 0 9 $ 656 0 $ 0 1 2 18 $ 1,312 Training 2 0 0 0 1 0 0 3 $ 174 0 $ 30 1 2 6 $ 408 Plan activities 16 0 0 0 0 0 0 16 $ 1,028 0 $ 30 1 2 32 $ 2,117 Procure engines 2 1 0 2 0 0 0 5 $ 288 0 $ 20 3 2 30 $ 1,846 Ship Engines 1 0 0 0 0 0 4 5 $ 214 0 $ 100 3 2 30 $ 1,883 Engine Maintenance 1 0 0 0 3 33 0 37 $ 1,309 0 $ 25 3 2 222 $ 8,006 Testing (

Inhouse 8 0 0 0 38 1 1 48 $ 2,310 0 $ 100 9 2 864 $ 43,388 Data entry and analysis 22 0 0 5 2 0 0 29 $ 1,660 0 1 3 2 174 9,968 Preparing and submitting report 18 1 1 1 0 0 0 21 $ 1,394 0 $ 3 1 2 42 $ 2,794 Store, file and maintain records 8 0 0 1 0 0 0 9 $ 545 0 $ 1 1 2 18 $ 1,093 Total per manufacturer 85 3 2 9 44 34 5 182 varies 0 varies varies N/ A N/ A N/ A Total for the industry N/ A N/ A N/ A N/ A N/ A N/ A N/ A N/ A $ 70,008 0 $ 2,804 N/ A 2 1,436 $ 72,812 ( 1) Includes diskettes, photocopying, postage expenses, phone calls, and testing costs, annualized.

See section 6( b)( iii) for details.

( 2) 1 = one time tasks 3 = tasks performed per engine family ( 3 engine families participate per manufacturer)

9 = number of tests performed/ engine manufacturer/ model year ( minimum of 3 tests per engine family participating in any given model year).

Refer to Section 6( d) for further detail.

average burden= total burden/# respond = 718 hrs per respon Annual Respondent Burden and Cost  Phase 2 Voluntary In useTesting Program Table 7 Hours and cost per application Total hours and cost Table 8 Annual Respondent Burden and Cost  Large Si In useTesting Program Hours and cost per application Total hours and cost Information Collection Activity Engineer @ 64.28/ hr Manager @

$ 96.96/ hr Legal @

$ 108.84/ hr Clerical@

$ 31.06/ hr Test Cell Operator@$

45.40/ hr Assembler @ $ 33.60/ hr Transport @

$ 37.88/ hr Respon.

hr/ yr Labor Cost/ yr Capital Startup Cost O & M Cost( 1) Frequency ( 2) Number of Respon. Total hr/ yr Total Cost/ yr Review of instructions and regulations 7 1 1 0 0 0 0 9 $ 656 0 $ 1 1 10 90 $ 6,568 Training 2 0 0 0 1 0 0 3 $ 174 0 $ 30 1 10 30 $ 2,040 Plan activities 16 0 0 0 0 0 0 16 $ 1,028 0 $ 30 1 10 160 $ 10,585 Procure engines 2 1 0 2 0 0 0 5 $ 288 0 $ 20 3 10 150 $ 9,229 Ship Engines 0 0 0 1 0 0 4 5 $ 183 0 $ 150 3 10 150 $ 9,977 Engine Maintenance 1 0 0 0 3 33 175 212 $ 7,938 0 $ 200 3 10 6,360 $ 244,148 Testing (

Inhouse 8 0 0 0 38 1 1 48 $ 2,311 0 $ 100 6.4 10 3,072 $ 154,299 Data entry and analysis 22 0 0 5 2 0 0 29 $ 1,660 0 $ 1 3 10 870 $ 49,838 Preparing and submitting report 18 1 1 1 0 0 0 21 $ 1,394 0 $ 3 1 10 210 $ 13,969 Store, file and maintain records 8 0 0 1 0 0 0 9 $ 545 0 $ 1 1 10 90 $ 5,463 Total per manufacturer 84 3 2 10 44 34 180 357 $ 16,177 0 varies varies N/ A N/ A N/ A Total for the industry N/ A N/ A N/ A N/ A N/ A N/ A N/ A N/ A $ 487,936 0 $ 18,180 N/ A 10 11,182 $ 506,116 ( 1) Includes diskettes, photocopying, postage expenses, phone calls, fuel and testing costs, annualized.

See section 6( b)( iii) for details. Average burden= total burden/# respond ( 2) 1 = one time tasks = 1,118 hrs per respon 3 = tasks performed per engine family ( 3 engine families participate per manufacturer)

9 = number of tests performed/ engine manufacturer/ model year ( minimum of 3 tests per engine family participating in any given model year).

Refer to Section 6( d) for further detail. Information Collection Activity Engineer @ 64.28/ hr Manager @

$ 96.96/ hr Legal @

$ 108.84/ hr Clerical@

$ 31.06/ hr Test Cell Operator @$ 45.40/

hr Assembler @

$ 33.60/ hr Transport @

$ 37.88/ hr Respon.

hr/ yr Labor Cost/ yr Capital Startup Cost O & M Cost( 1) Frequency ( 2) Number of Respon.

( 3) Total hr/ yr Total Cost/ yr Provide Pre audit information 5 2 0 1 0 0 0 8 $ 546 0 $ 0 1 7 56 $ 3,825 Review of instructions and regulations 7 1 1 0 0 0 0 9 $ 656 0 $ 0 1 4 36 $ 2,623 Training 3 1 0 0 1 0 0 5 $ 335 0 $ 30 1 4 20 $ 1,461 Plan activities 16 0 0 0 0 0 0 16 $ 1,028 0 $ 30 1 4 64 $ 4,234 Testing ( Production < 100K engines)

In house 8 0 0 0 38 1 1 48 $ 2,311 0 $ 100 2 1 96 $ 4,822 Testing ( Production < 100K engines)

Contract Out 0 0 0 0 0 0 0 0 $ 0 0 $ 2,750 2 1 0 $ 5,500 Testing ( Production 100k+ engines)

Inhouse 8 0 0 0 38 1 1 48 $ 2,311 0 $ 100 4 2 384 $ 19,287 Data entry and analysis 22 0 0 5 2 0 0 29 $ 1,660 0 1 1 4 116 6,645 Preparing and submitting report 18 1 1 1 0 0 0 21 $ 1,394 0 $ 3 1 4 84 $ 5,588 Store, file and maintain records 8 0 0 1 0 0 0 9 $ 545 0 $ 1 1 4 36 $ 2,185 Total per manufacturer 95 5 2 8 79 2 2 193 varies 0 varies varies N/ A N/ A N/ A Total for the industry N/ A N/ A N/ A N/ A N/ A N/ A N/ A N/ A $ 49,409 0 $ 6,760 N/ A 7 892 56,169 ( 1) Includes diskettes, photocopying, postage expenses, phone calls, and testing costs, annualized.

See section 6( b)( iii) for details.

( 2) 1 = one time tasks; other # = number of tests performed/ eng manufacturer/ model year Refer to Section 6( d) for further detail.

( 3) EPA estimates it will audit 12 manufacturers over the next 3 years ( 4 annually). It might, however, request pre audit information from approximately 7 manufacturers each year.

average burden= total burden/# respond = 127.428571 hrs per respon Table 9 Annual Respondent Burden and Cost  Selective Enforcement Auditing Program Hours and cost per application Total hours and cost Information Collection Activity Engineer @$ 62.66/ hr Senior @

$ 17.65 Attorney @

$ 66.34/ hr Manager @

$ 76.85/ hr SES 1 @

135.15 Respon.

hr/ yr Labor cost/ yr Capital Startup Cost O & M Cost ( 1) Applications / respon Number of Respon. Total hr/ yr Total cost/ yr Review of regs and standards 2 1 4 3 1 11 $ 774 0 $ 2 1 106 1,166 $ 82,259 Entering data from applications into database 1 1.75 0 0 0 2.75 $ 94 0 $ 0 16 106 4,664 $ 158,657 Reviewing applications,

asking questions 7 3 0 0 0 10 $ 492 0 $ 10 16 106 16,960 $ 850,663 Verifying that the correct engines have been tested 0.5 0.5 0 0 0 1 $ 40 0 $ 0 16 106 1,696 $ 68,103 Answering manufacturers'

questions 4 2.5 3 2 1 12.5 $ 783 0 $ 10 16 106 21,200 $ 1,344,309 Issuing appropriate certificates 1 0.5 0 0 0 1.5 $ 71 0 $ 2 16 106 2,544 $ 124,631 Storing data 1 2 0 0 0 3 $ 98 0 $ 8 16 106 5,088 $ 179,708 Answering questions from the public 2 5 3 3 1 14 $ 778 0 $ 10 16 106 23,744 $ 1,336,940 Upgrading and maintaining the database 80 10 0 10 2 102 $ 6,228 0 $ 5 1 1 102 $ 6,233 Handling requests for confidentiality 5 1 2 1 1 10 $ 676 0 $ 3 1 1 10 $ 679 Making information available to the public 10 3 0 1 1 15 $ 892 0 $ 5 1 1 15 $ 897 Total 113.5 30.25 12 20 7 182.75 $ 10,925 0 $ 55 N/ A N/ A N/ A N/ A Total for the agency N/ A N/ A N/ A N/ A N/ A N/ A $ 4,085,012 0 $ 68,065 16 106 77,189 4,153,077 Table 11 Annual Agency Burden and Cost  Certification Program SI Engines and Recreational Vehicles Hours and cost per application Total hours and cost ( 1) Includes photocopying, postage expenses and calls. Table 12 Annual Agency Burden and Cost  Average, Banking and Trading Program Hours and cost per application Total hours and cost Information Collection Activity Engineer @$ 62.66/ hr Respon.

hr/ yr Labor cost/ yr Capital Startup Cost O & M Cost ( 1) Number of Respon. Total hr/ yr Total cost/ yr Review regulations 16 16 1,003 0 5 1 16 1,008 Answer respondent questions 2 2 125 0 5 31 62 4,040 Review submitted reports 2 2 125 0 1 31 62 3,916 Enter data from reports into database 0.5 1 31 0 1 31 16 1,002 Audit reports and records 5 5 313 0 0 31 155 9,712 Recordkeeping 0.5 1 31 0 2 31 16 1,033 Total per manufacturer 26 26 1,629 0 14 N/ A N/ A N/ A Total for the industry N/ A N/ A $ 20,427 0 $ 284 31 326 $ 20,711 ( 1) Includes photocopying and postage expenses.

SI Engines and Recreational Vehicles Table 13 Annual Agency Burden and Cost  Production Line Testing Program Information Collection Activity Engineer @$ 62.66/ hr Attorney @

$ 66.34/ hr Manager @$ 76.85/ hr Agency hr/ yr Agency Labor cost/ yr Capital Startup Cost O & M Cost ( 1) Frequency Number of Respon. Total hr/ yr Total cost/ yr Review regulations 7.0 3.0 1.0 11 $ 714 0 $ 1 1 1 11 $ 715 Answer respondent questions 5.0 1.0 2.0 8 $ 533 0 $ 5 1 69 552 $ 37,145 Review reports for format and completeness 1.0 0.0 0.0 1 $ 63 0 $ 0 4 69 276 $ 17,294 Analyze reports 4.0 0.0 0.0 4 $ 251 0 $ 0 1 69 276 $ 17,294 Request additional information, if needed 2.0 0.0 1.0 3 $ 202 0 $ 5 4 69 828 $ 57,179 Enter data from reports into database and file submittals 0.5 0.0 0.0 1 $ 31 0 $ 1 4 69 138 $ 8,923 Maintain/ enhance database 8.0 0.0 1.0 9 $ 578 0 $ 0 1 1 9 $ 578 Post data in Internet 3.0 0.0 0.0 3 $ 188 0 $ 0 1 1 3 $ 188 Analyze requests for confidentiality 4.0 3.0 1.0 8 $ 527 0 $ 1 1 1 8 $ 528 Audit reports and records 5.0 0.0 1.0 6 $ 390 0 $ 3 1 1 6 $ 393 Total per participating engine family 40 7 7 54 $ 3,477 0 $ 16 varies 10 N/ A N/ A Total for the agency N/ A N/ A N/ A N/ A $ 138,232 0 $ 2,006 N/ A 10 2,107 $ 140,238 ( 1) Includes photocopying, postage expenses and calls. SI Engines and Recreational Vehicles Hours and cost per engine family Table 14 Annual Agency Burden and Cost  In use Testing Program Hours and cost per application Total hours and cost Information Collection Activity Engineer @

$ 62.66/ hr Attorney @

$ 66.34/ hr Manager @

$ 76.85/ hr Agency hr/ yr Labor cost/ yr Capital Startup Cost O & M Cost ( 1) Number of Respon. Total hr/ yr Total cost/ yr Review Regulations 20.0 2.0 1.0 23 $ 1,463 0 0 1 23 1,463 Answer respondent questions 3.0 1.0 1.0 5 331 0 7 51 255 17,247 Evaluate testing plans 3.0 0.0 1.0 4 265 0 2 51 204 13,608 Enter data from reports into database 1.0 0.0 0.0 1 63 0 0 51 51 3,196 Review submitted reports for format and completeness 0.5 0.0 0.0 1 31 0 2 51 26 1,700 Analyze reports 5.0 0.0 0.0 5 313 0 0 51 255 15,978 Request additional information if needed 1.0 0.0 1.0 2 140 0 5 51 102 7,370 Analyze requests for confidentiality 1.0 1.0 1.0 3 206 0 1 51 153 10,549 Maintain/

enhance database 10.0 0.0 0.0 10 627 0 0 1 10 627 Total 45 4 54 3,438 0 17 N/ A N/ A N/ A Total for the agency N/ A N/ A N/ A $ 70,870 0 $ 867 51 1,079 $ 71,737 ( 1) Includes photocopying, postage expenses and calls.

SI Engines and Recreational Vehicles Information Collection Activity Division Director SES @ $ 135.15/ hr Group Manager GS 15 @ $ 76.85 Engineer @$ 62.66/ hr Attorney @

$ 66.34/ hr Agency hr/ yr Labor cost/ yr Capital Startup Cost O & M Cost ( 1) Number of Respon. Total hr/ yr Total cost/ yr Review Regulations 1 3 14 2 20 1,376 0 5 1 20 1,381 Answer respondent questions 0 5 14 1 20 1,328 0 10 7 140 9,365 Select candidates 0 1 10 0 11 703 0 3 1 11 706 Request pre audit data 0 1 6 1 8 519 0 5 7 56 3,669 Final selection of manufacturers to be audited 0.5 0.5 2 0 3 231 0 0 1 3 231 Issue test orders 1 2 4 1 8 606 0 5 4 32 2,443 Perform audit/ witness testing 0 0 80 0 80 5,013 0 5,000 4 320 40,051 Enter data from reports into database 0 0 2 0 2 125 0 0 4 8 501 Review reports for completeness and file 0 0 2 0 2 125 0 0 4 8 501 Analyze reports 0 0 8 0 8 501 0 0 4 32 2,005 Total per manufacturer 2.5 12.5 142 5 162 10,528 0 5,028 varies N/ A N/ A Total for the agency N/ A N/ A N/ A N/ A N/ A $ 40,721 0 $ 20,133 7 630 $ 60,854 ( 1) Includes cost of traveling to testing site, photocopying, postage expenses and calls. Table 15 Annual Agency Burden and Cost  Selective Enforcement AuditingProgram SI Engines and Recreational Vehicles Hours and cost per respondent Total hours and cost