Goshen CSD - Updated Service Agreement (Proposed) — Visalia City Council, June 15, 2026

REVISED AND RESTATED WASTEWATER SERVICE AGREEMENT between the City of Visalia and the Goshen Community Services District THIS AGREEMENT is made and entered into as of this day of , 2026, by and between the GOSHEN COMMUNITY SERVICES DISTRICT, a special district formed under the Government Code 6100 (hereinafter called "District"), and the CITY OF VISALIA, a Charter city created and existing under laws of the State of California (hereinafter called "City"). The District and the City may be referred to herein as the “Parties”. RECITALS A. WHEREAS, the District has the power and authority to provide for collection, transmission, treatment and disposal of wastewater and is authorized to contract for such services; and, B. WHEREAS, the District has constructed a wastewater collection and transmission system for the community of Goshen which collects wastewater within the jurisdictional boundaries of the District that requires adequate treatment and disposal; and, C. WHEREAS, the City owns and operates conveyance facilities, a wastewater treatment and disposal facility (hereinafter called "Plant"), located at 7579 Avenue 288, Visalia, California 93277, with the capability of accepting, treating and disposing of the wastewater proposed to be collected and discharged from the District to comply with the standards required by the Regional Water Quality Control Board or other authority; and, D. WHEREAS, the City is willing to accept District’s collected wastewater within the limits set forth in this Agreement and to provide treatment and disposal services for the collected wastewater, and is authorized to do so under applicable law, subject to the provisions of this Agreement. E. WHEREAS, the Parties have been operating pursuant to the terms of a Wastewater Service Agreement dated June 5, 1995, and desire to replace that agreement with this Revised and Restated Wastewater Service Agreement. NOW, THEREFORE, in consideration of the foregoing and other good and valuable consideration, the receipt and sufficiency of which is acknowledged by all parties hereto, it is agreed that the Wastewater Service Agreement dated June 5, 1995 is hereby repealed and is of no further force and effect, and that this Revised and Restated Wastewater Service Agreement fully states the rights and obligations of the parties with respect to the acceptance by the City of wastewater collected by the District, as follows: ARTICLE I. ACCEPTANCE AND TREATMENT OF WASTEWATER. 1.1. Wastewater Acceptance and Discharge. The City agrees to accept, treat and dispose of, and the District agrees to discharge all sanitary and process wastewater it collects from the District, into the sewerage system of the City for treatment and disposal at the Plant, subject to the limitations and terms and conditions set forth herein.

1.2. Connection to City Collection System. Connection from District to the City shall be through a twenty four (24) inch gravity sewer line (“District Main Line”) that has been constructed and operated by the District in Camp Drive, which conveys collected wastewater to connect to the existing City Highway 198-Airport lift station (as depicted on the attached Exhibit “A”) Notwithstanding previous agreements between the Parties to the contrary, the City and District agree that the District Main Line is and shall remain the property of the District, and District shall be solely liable for the maintenance of the District Main Line. City is and will remain responsible for any improvements to its lift station and conveyance facilities downstream of the point of connection. The District Main Line was planned to provide full capacity for the ultimate build out of the Goshen sphere of influence. Should the line capacity have to be increased or replaced in the future, such increases or replacement shall be the responsibility of District. ARTICLE II. PARAMETERS OF DISCHARGE 2.1 Discharge V olume and Loading Limits. 2.1.1 Average Daily Discharge: On an annual basis, the District’s average daily discharge shall not exceed the following limits: Average Daily Parameters (Annual Average) Wastewater Flow (“Flow”) 563,000 gallons per day Biochemical Oxygen Demand (“BOD”) 1,750 pounds per day Suspended Solids (“SS”) 1,460 pounds per day 2.1.2 Maximum Daily Discharge: District’s maximum daily discharge shall not exceed any of the following limits: Maximum Daily Parameters Wastewater Flow (“Flow”) 850,000 gallons per day Biochemical Oxygen Demand (“BOD”) 2,600 pounds per day Suspended Solids (“SS”) 2,200 pounds per day 2.2 Other Limitations: The wastewater discharge into the City system shall not: 2.2.1 Contain oils, toxic chemicals, diatomaceous earth or other biologically toxic materials, as more specifically described in Sections 2.2.2 through 2.2.4 below, which would interfere with wastewater treatment processes or adversely affect the quality of the Plant's effluent. 2.2.2 Have a pollutant level that causes the City's effluent from the Plant to exceed the requirements of the Regional Water Quality Control Board. In the event that the District's discharge causes the plant to exceed the requirements of the Regional Water Control Board thereby causing the City to modify its operations to comply with such requirements, the District, at its own option, shall either modify the loading from the District to achieve compliance or pay the City the actual cost of treating the noncomplying loading provided that: (i) the City can establish to the reasonable satisfaction of the District that the City cannot comply with such discharge requirements unless the District adjusts the loading levels of its discharge from the District, (ii) such adjustment required of the District is no more than the minimum necessary to enable the City to comply with discharge requirements, and (iii) following any such adjustment all then existing and future dischargers to the Plant are subject to no less restrictive pollutant limitations than the District. 2.2.3 Have an average daily wastewater temperature in excess of 150°F. 2.2.4 Violate the Environmental Protection Agency pretreatment requirements applicable to the City as administered by the Regional Water Quality Control Board. 2.3 Preliminary Treatment Standards: The District shall be responsible for adopting and implementing a pretreatment program that identifies exceedances from individual discharges within the District jurisdictional boundaries and collection system and requires pretreatment to avoid continued exceedances. The District’s pretreatment program shall be consistent with City and state and federal Environmental Protection Agency best practices and standards. The District will submit all reports and pretreatment program documents to the City, and as the Regional Water Quality Control Board permit holder, the City shall be solely responsible for incorporating and submitting the District pretreatment program data and information to the Regional Water Quality Control Board. 2.4 Enforcement of District Discharge Requirements. As part of the District pretreatment program, Goshen shall maintain and implement an enforcement response plan for enforcing internal District discharge requirements applicable to discharges to District’s collection system. 2.5 Representations Regarding Capacity. 2.5.1 The City acknowledges and agrees that the District has purchased the right to discharge wastewater to the City system in the amounts specified in Section 2.1 above, subject to ongoing payment of the service fees and charges therefor as provided in Article III herein, and that the District has a vested right to discharge such wastewater at any time, continuously and from time to time, subject to the provisions set forth herein. 2.5.2 The District acknowledges that the City has constructed its collection, conveyance and treatment facilities in a manner that relies on the limitations set forth in this Agreement, and unless otherwise provided in this Agreement has not constructed its collection, conveyance and treatment facilities in a manner to accommodate for unplanned increases in the discharge flow and load limitations above those set forth in this Agreement. Subject to the foregoing acknowledgment, the City has advised the District that the City has capacity in its existing City system which will facilitate treatment of the effluent to be discharged from the District pursuant to this Agreement. The City warrants, represents and agrees that at all times beginning with the District's commencement of operations within the District through the duration of this Agreement, City will make its best efforts to accept for treatment under applicable laws and regulations of Federal, state, regional and local authority any and all discharges from the District within the parameters set forth in Section 2.1, the increased discharges set forth in Article IV, and any additional future discharges as are mutually agreeable between the parties. It is anticipated that the current District infrastructure and collection system is sufficient for the District’s sphere of influence build-out, but the District shall be responsible for the costs of constructing and installing any and all sewer line(s) from District's collection system, and for any flow meters, automated sampling, odor control and connection to the City system if necessary under this Agreement. 2.5.3 City shall not restrict, limit or otherwise mandate to the District in its determination of allocating the purchased capacity to any other person or entity, existing or proposed, within the adopted District Boundary, as may be amended from time to time, or within the District's Sphere of Influence as approved by the Tulare County Local Agency Formation Commission, except when service to such person or entity would impair, or adversely affect the ability of the conveyance facilities or the Plant to accept and treat the District's wastewater as provided herein beyond normal capacity, treatment and disposal impacts. The District agrees to make a good faith effort to notify the City of any potential increases in wastewater flow, biochemical oxygen demand, suspended solids and other potential pollutant levels, indicated by any commercial and/or industrial development inquiries, that would significantly affect the quantity and/or quality of the District's discharge to the City system as soon as such potential impacts are made known to the District. Such notification is limited to the anticipated effect on the system and the anticipated change in loadings and does not include specifics regarding those persons or entities making the inquiry. 2.5.4 In relation to any proposed modification of the Goshen Community Plan, the Goshen Sphere of Influence or the District boundaries, District shall advise the Tulare County Local Agency Formation Commission and County of Tulare, including the Resource Management Agency and the Tulare County Planning Commission, that its ability to discharge wastewater to the City is limited by the terms of this Agreement. 2.5.5 The City shall not contract, agree or otherwise create wastewater collection, treatment and disposal service with any entity, corporation or individual which resides, does business within or requests service for any parcel, building, street or property within the boundary of the District. The City shall not renew any current contract with any entity, corporation, industry or property for wastewater service within the District at expiration thereof. 2.6 Determination of BOD and Suspended Solids Levels. In order to determine the BOD and suspended solids characteristics of the District's wastewaters, the Parties agree as follows: 2.6.1 Sampling. The District, at the District's cost, shall maintain and operate a wastewater sampler and recording meter, with chart, which is capable of collecting 24-hour composite samples and accurately sampling the wastewater flow in proportion to the volume thereof. The District shall maintain and operate, at District's cost, and in a location satisfactory to City, a continuous recording pH meter. District shall maintain said pH meter at District's cost during the term of this Agreement. The parties acknowledge that the flow meter, pH meter and sampler are presently installed in a location satisfactory to the parties hereto and the District agrees to maintain such meters and sampler at District's cost. City shall be responsible for the collection of the samples and recordings at such intervals and for such durations as determined by the City. The District may monitor the sampling procedure and the samples collected and stored. At the end of each required sampling period, the sample shall be well mixed and divided equally between City and District. District shall provide City access to said sampling and recording equipment so that City may, at any reasonable time, collect and analyze samples and recordings. 2.6.2 Analyses of Samples. City may analyze the samples for the purpose of determining the BOD and suspended solids characteristics of District's wastewater discharge as may be relevant. The cost of such analysis shall be included and part of the monthly O & M Charge paid by the District, and not an additional charge to the District. All analyses of samples shall be made in accordance with the then current edition of "Standard Methods for the Examination of Water and Wastewater" or such standard reasonably acceptable to both parties, if the foregoing publication shall be discontinued. In the event of exceedances requiring multiple resampling to confirm correction of exceedances, District shall be responsible for the cost of repeated samples, which shall be invoiced to the District by the City as part of monthly service charges. 2.6.3 Evaluation of Loadings. The results of the analyses of samples shall be treated in accordance with the procedures set forth in Exhibit "B" attached hereto and shall be the basis for establishing monthly billing. The City shall provide the District with copies of all flow loadings and sample test results, upon request. 2.7 Right to Reclaimed Water. The City shall have the sole right to the use and disposition of any reclaimed water that is the product of the City’s treatment of discharge wastewater received from District. The right to use of reclaimed water includes any right to credits or offsets that may be recognized by a Groundwater Sustainability Agency in the implementation of a Groundwater Sustainability Plan under the California Sustainable Groundwater Management Act. District shall not attempt to claim any such credits or offsets on its own behalf or on behalf of any other person, entity or agency. ARTICLE III. SERVICE CHARGES 3.1 Ongoing Sewer Service Charges. The District shall pay to the City monthly sewer service charges in accordance with rates established by City Resolution. The Sewer Service Charges shall consist of O & M Charge as defined by City Resolution. Nothing in this Agreement shall prevent the City from amending its rate schedule for such Sewer Service Charges from time to time, provided that any such charges to the District shall be consistent with the then current rates and charges in effect for other customers with similar sewer service demands and the provisions of Section 204(b)(l)(A) of the Federal Water Pollution Control Act, 33 U.S.C § 1284(b)(l)(A), and any guidelines adopted by the Environmental Protection Agency in accordance therewith, and provided further that in any event such amendments shall not discriminate against the District. Notwithstanding the foregoing, City reserves the right to establish a separate cost based upon a rate study specific to meeting and handling Goshen CSD effluent demands, provided the basis and methodology used to determine cost recovery is the same as applied to other City sewer customers with similar sewer service demands, and provided Goshen CSD is provided notice and opportunity to comment on any proposed specific rate study. District will be responsible for processing any adjustment of its rates applicable to District’s customers that may be necessary to pay for any adjusted City Sewer Service Charge, and District and City shall coordinate their respective public review processes to the greatest extent feasible to ensure the City’s Sewer Service Charge and the District’s rate adjustments are processed simultaneously. Any rate increase in any Sewer Service Charge established by resolution of the City to be imposed upon the District shall be effective upon approval by the District of adjusted customer rates, or within six (6) months after City provides District with written notice of the approved adjusted Sewer Service Charge, whichever is earlier. 3.2 Penalties. Any exceedance of the Section 2.1 quantities (as may be amended per terms of this Agreement), or discharges of forbidden substances as per Section 2.2.1, will be assessed penalties calculated at the same rate as Significant Industrial Users, as set forth in duly adopted City ordinances or rules and regulations, or as may be set forth in a Goshen CSD-specific rate study. Assessment of penalties pursuant to this paragraph shall not be the sole remedy available to the City for addressing violations of this Agreement, and City reserves the right to seek additional remedies per paragraph 6.1 of this Agreement. 3.3 Payment. The City shall bill the District monthly for Ongoing Sewer Service Charges and the District shall pay the City within thirty (30) days upon receipt of said billing. ARTICLE IV . INCREASE IN DISCHARGE FLOW AND LOADING LIMITS 4.1 One Expansion of Capacities, in stages, based on 2024 Master Plan. City has completed an update to its Water Reclamation Facility - Master Plan, and pursuant to a request by the District, included in that update a planned increase in the flow and load limitations from Goshen CSD over those quantities provided in Section 2.1 of this Agreement. The Parties agree that District is obligated to increase the flow and load limitations and to pay the fees and charges on the schedule as set forth in this Article IV . 4.2 2027 Expansion: No earlier than 07/01/2026, and no later than 01/03/2028, District shall pay the applicable Capacity Purchase Fees as defined in Section 4.4 below, upon which the quantities provided in Section 2.1 above shall be deemed amended to be as follows: 4.2.1 2027 Amended Average Daily Discharge: On an annual basis, the District’s daily average discharge shall not exceed the following limits:

Average Daily Parameters (Annual Average) Wastewater Flow (“Flow”) 886,000 gallons per day Biochemical Oxygen Demand (“BOD”) 2,200 pounds per day Suspended Solids (“SS”) 2,300 pounds per day 4.2.2 2027 Amended Maximum Daily Discharge: District’s maximum daily discharge shall not exceed any of the following limits: Maximum Daily Parameters Wastewater Flow (“Flow”) 1,329,000 gallons per day Biochemical Oxygen Demand (“BOD”) 4,100 pounds per day Suspended Solids (“SS”) 3,400 pounds per day 4.3 2030 Expansion: No earlier than 01/01/2030, and no later than 06/03/2030, District shall pay the applicable Capacity Purchase Fees as defined in Section 4.4 below, upon which the quantities provided in Section 2.1 above shall be deemed amended to be as follows: 4.3.1 2030 Amended Average Daily Discharge: On an annual basis, the District’s daily average discharge shall not exceed the following limits: Average Daily Parameters (Annual Average) Wastewater Flow (“Flow”) 992,000 gallons per day Biochemical Oxygen Demand (“BOD”) 3,100 pounds per day Suspended Solids (“SS”) 2,600 pounds per day 4.3.2 2030 Amended Maximum Daily Discharge: District’s maximum daily discharge shall not exceed any of the following limits: Maximum Daily Parameters Wastewater Flow (“Flow”) 1,488,000 gallons per day Biochemical Oxygen Demand (“BOD”) 4,600 pounds per day Suspended Solids (“SS”) 3,800 pounds per day 4.4 Capacity Purchase Fees. As a condition to the expanded capacities as provided in Sections 4.2 and 4.3 above, District shall pay a Capacity Purchase Fee at the time of each increase, determined by applying the following fees to the difference between the limitation prior to increase and the limitation after the increase: 4.4.1 A Treatment Plant Connection Capacity Charges, applied and calculated based on the Average Daily Parameters for flow and pounds per day for BOD and SS, at the same amount as would be applied to development within the City as a development impact fee; and 4.4.2 A Trunk Line Capacity Charge, applied and calculated based on the Maximum Daily Parameters for flow and pounds per day for BOD and SS, at the same amount as would be applied to development within the City as a development impact fee. The Capacity Purchase Fee will be calculated by applying the identified development impact fee in place at the time of payment of the fee. District acknowledges that the City schedule of impact fees, including those applicable to this Agreement, is reviewed and adjusted annually on or about July 30 of each year. Further, District acknowledges that a comprehensive review of the current schedule of impact fees, including the Treatment Plant Connection Capacity Charge and the Trunk Line Capacity Chart, is underway and anticipated to be adopted prior to the end of calendar year 2026. District shall have the right to review and comment on any revision to the impact fees as they will be applied to capacity expansions as provided in this Article IV . District may purchase the capacity increases in stages, in which case the Capacity Purchase Fee for the portion purchased shall be based on the fee schedule then in effect, and District will have access only to that portion of capacity increases purchased and paid for. The full capacity increases must be purchased and paid for within the time frames described in Sections 4.2 and 4.3 above. City represents that the current Treatment Plant Connection Capacity Charge and Trunk Line Capacity Charge that have been in effect as of July 30, 2025, are as described in Exhibit “C”, which is incorporated herein by this reference. City further represents that such charges will remain in effect until July 30, 2026. To demonstrate the application of the Treatment Plant Connection Capacity Charge and Trunk Line Capacity Charge, the City has calculated the Capacity Purchase Fee that would be payable in the event District elects to purchase and pay for the full amount of the 2027 Expansion prior to July 30, 2026, and such calculation is shown in Exhibit “C”. 4.5 No Additional Expansion Rights. Other than as set forth herein, District will not have any right to expand the capacities provided for in this Agreement, except as otherwise may be mutually agreeable between the parties and evidenced in writing. District acknowledges that the current Water Reclamation Facility Master Plan accommodates only those amounts of flow and loading discharges committed to in this Agreement. City does not anticipate a further Master Plan update for at least the next 10 years. As a result, City does not believe that it will have any ability to accept additional discharges from the District beyond the planned 2027 and 2030 capacity increases. In the event District determines that it foresees the need for additional capacity beyond those amounts provided in this Agreement, prior to the City studying and updating the Water Reclamation Facility Master Plan, the District will be solely responsible for paying for the City’s actual costs, including consultant and engineer costs, in identifying, planning and financing any upgrades to the City conveyance and treatment facilities that may be required to create the additional capacity. City further makes no representation or warranty that the planning process will be successful in identifying feasible upgrades of conveyance or treatment capacities, and does not commit to the use of Capacity Purchase Fees as an appropriate mechanism for the District to pay for the cost of any future conveyance or treatment upgrades that may be identified as being feasible. The City shall provide the District with written notice whenever the City has been authorized to study and update the Water Reclamation Facility Master Plan, which is not at the specific request of the District, and the parties shall work cooperatively and in good faith to identify upgrades that would allow further expansion of capacity to the District. ARTICLE V . CONTRACT TERM 5.1 Term. This agreement shall continue until terminated pursuant to Section 5.2. 5.2 Termination. This agreement may only be terminated upon the written consent of all parties. Such consent may be withheld in the sole discretion of each affected party. ARTICLE VI. REMEDIES 6.1 Specific Performance. The parties hereto recognize that since the extent of damages caused by any breach of the provisions of this Agreement may be extremely difficult to determine, and any action for damages may be an inadequate remedy for such breach, an action for specific performance may be necessary to provide an adequate remedy for such breach, and it is agreed that nothing contained herein is intended to make unavailable, the remedy of specific performance. Without limiting the foregoing, it is expressly acknowledged and agreed that District would suffer irreparable harm and injury, for which damages or other remedy at law would not be adequate, should the City in any manner seek or take any action to impair or reduce City's ability to accept any wastewater discharges from the District up to the discharge limits set forth in Article II above. District and City expressly recognize and agree that in such instance, and without limiting any other remedies available to District, District shall be entitled to pursue injunctive relief, restraining order, or other appropriate remedies to prevent City from depleting or in any way reducing its ability to accept for treatment any of District's wastewater discharges in accordance with the terms of this Agreement and/or to require City to take such actions as may be necessary to accept such wastewater discharges for treatment. Also without limiting the foregoing, it is expressly acknowledged and agreed that the City would suffer irreparable harm and injury, for which damages or other remedy at law would not be adequate, should the District seek or take any action, including but not limited to discharge in a manner in violation with any statute, ordinance, or other state or federal recommendation or law, or limitations of this Agreement, or fail to enforce any pre-treatment limits. District and City expressly recognize and agree that in such instance, and without limiting any other remedies available to City, City shall be entitled to pursue injunctive relief, restraining order, or other appropriate remedies and/or to require District to take such action(s) as may be necessary. 6.2 Force Majeure. Notwithstanding anything to the contrary herein, it is agreed that in the event and to the extent that fire, explosion, stoppage of labor, war, act of God, pandemic, act of the public enemy, accident, strike, labor troubles (whether or not within the power of the party to settle the same), judgment, decree or order of a competent judicial or governmental authority, or any other sources whatsoever beyond the control of a party hereto (excluding a financial inability to perform), whether similar or dissimilar to the causes mentioned, prevents or delays performance by a party hereto. that party shall be relieved of such performance and the consequences thereof, without liability, for so long as and to the extent that such performance is prevented by such cause; provided, however, that the party excused from such performance shall use due diligence in resuming same at the earliest practical time, and provided further that any suspension of the services to be provided by City to District hereunder and any restoration of such suspended services shall not be made in any manner which discriminates against District in relation to any other users of the Plant. ARTICLE VII. HOLD HARMLESS 7.1 City. The City shall hold harmless, defend and indemnify the District, their agents, officers and employees from and against any liability, claims, actions, costs, damages or losses of any kind, including death or injury to any person and/or damage to property, including District property, arising out of the activities of the City or its agents, officers and employees under this Agreement. This indemnification obligation shall continue beyond the term of this Agreement as to any acts or omissions occurring under this Agreement or any extension of this Agreement. 7.2 District. The District shall hold harmless, defend and indemnify the City, their agents, officers and employees from and against any liability, claims, actions, costs, damages or losses of any kind, including death or injury to any person and/or damage to property, including City property, arising out of the activities of the District or its agents, officers and employees under this Agreement. This indemnification obligation shall continue beyond the term of this Agreement as to any acts or omissions occurring under this Agreement or any extension of this Agreement. ARTICLE VIII. MISCELLANEOUS 8.1 Waiver. No waiver by City or District of any term or condition of this Agreement shall be deemed to be or construed as a waiver of any other term or condition, nor shall a waiver of any breach be deemed to constitute a waiver of any subsequent breach, whether of the same or a different provision of this Agreement.

8.2 Assignment. No party hereto shall have the right to assign this Agreement or any of its rights and obligations hereunder without first securing the other party's written consent, except that this prohibition shall not be applicable to any change of ownership of the District sewer system improvements and/or operation and maintenance involving the District. Such consent shall not unreasonably be withheld or delayed. 8.3 Notices. Whenever notice is required or permitted hereunder from one party to the other, the same shall be in writing and shall be given effect by hand delivery or by mailing same by certified or registered mail, to the party to whom given as follows: City: City of Visalia Attention: City Manager 220 N. Santa Fe St. Visalia, California 93291 District: Goshen Community Services District Attention: General Manager P.O. Box 2 Goshen, California, 93227 or to such other person or address as may hereinafter be designated by one party to the other in writing. Notice by certified or registered mail shall be deemed to have been given as of the date it is mailed, postage prepaid. 8.4 Changes. This Agreement cannot be changed, discharged, or terminated orally, but only by agreement in writing, signed by the parties hereto. 8.5 Law. This Agreement shall be deemed to be made, entered into and executed in, and shall be construed in accordance with the laws of the State of California. Venue is only appropriate in either Tulare County or the Eastern District of the United States District Courts. 8.6 Construction. Whenever possible, each provision of this Agreement shall be interpreted in such manner as to be effective and valid under applicable law, but if any provision of this Agreement shall be prohibited or invalid under applicable law, such provision shall be ineffective to the extent of such prohibition or invalidity without invalidating the remainder of such provision or any other provision of this Agreement. 8.7 Headings. Article and Section headings in this Agreement are for convenience only and are not to be construed as a part of this Agreement or in any way limiting or amplifying the provisions hereof. 8.8 Attorney's Fees. If any legal action or other proceeding is brought for the enforcement of this Agreement, or because of any alleged dispute, breach, or default arising out of or in connection therewith, the successful or prevailing party shall be entitled to recover reasonable attorneys' fees and other costs and expenses incurred in connection with such action or proceeding in addition to any other relief to which it may be entitled.

8.9 Entirety. This Agreement merges and supersedes all prior negotiations, representations and preliminary or other agreements between the parties hereto relating to the subject matter hereof, and constitutes the entire contract between the City and District for the acceptance and treatment of wastewater from the District. This Agreement shall control in the event of any conflict or inconsistency with any other provisions of any City Ordinance or any rules, resolutions, or regulations incorporated therein or adopted pursuant thereto. 8.10 Counterparts. This Agreement may be executed in one or more counterparts, and all so executed shall constitute one contract, binding on the parties, notwithstanding that all parties are not signatory to the same counterpart. IN WITNESS WHEREOF, the parties hereto have caused this Agreement to be executed as of the date first herein written. GOSHEN COMMUNITY SERVICE DISTRICT _______________________________________ Stephen Palermo, Board President CITY OF VISALIA _______________________________________ Leslie Caviglia, City Manager Exhibit "A"
5210 BIOCHEMICAL OXYGEN DEMAND (BOD)*
5210 A. Introduction

1. General Discussion
The biochemical oxygen demand (BOD) determination is an empirical test in which standardized laboratory procedures are used to determine the relative oxygen requirements of wastewa- ters, efﬂuents, and polluted waters. The test has its widest ap- plication in measuring waste loadings to treatment plants and in evaluating the BOD-removal efﬁciency of such treatment sys- tems. The test measures the molecular oxygen utilized during a speciﬁed incubation period for the biochemical degradation of organic material (carbonaceous demand) and the oxygen used to oxidize inorganic material such as sulﬁdes and ferrous iron. It also may measure the amount of oxygen used to oxidize reduced forms of nitrogen (nitrogenous demand) unless their oxidation is prevented by an inhibitor. The seeding and dilution procedures provide an estimate of the BOD at pH 6.5 to 7.5.

Measurements of oxygen consumed in a 5-d test period (5-d BOD or BOD
5, 5210B), oxygen consumed after 60 to 90 d of
incubation (ultimate BOD or UBOD, 5210C), and continuous oxygen uptake (respirometric method, 5210D) are described here. Many other variations of oxygen demand measurements exist, including using shorter and longer incubation periods and tests to determine rates of oxygen uptake. Alternative seeding, dilution, and incubation conditions can be chosen to mimic receiving-water conditions, thereby providing an estimate of the environmental effects of wastewaters and efﬂuents.

The UBOD measures the oxygen required for the total degra- dation of organic material (ultimate carbonaceous demand) and/or the oxygen to oxidize reduced nitrogen compounds (ul- timate nitrogenous demand). UBOD values and appropriate ki- netic descriptions are needed in water quality modeling studies such as UBOD:BOD
5 ratios for relating stream assimilative
capacity to regulatory requirements; deﬁnition of river, estuary, or lake deoxygenation kinetics; and instream ultimate carbona- ceous BOD (UCBOD) values for model calibration.

2. Carbonaceous Versus Nitrogenous BOD
A number of factors, for example, soluble versus particulate organics, settleable and ﬂoatable solids, oxidation of reduced iron and sulfur compounds, or lack of mixing may affect the accuracy and precision of BOD measurements. Presently, there is no way to include adjustments or corrections to account for the effect of these factors.

Oxidation of reduced forms of nitrogen, such as ammonia and organic nitrogen, can be mediated by microorganisms and exert nitrogenous demand. Nitrogenous demand historically has been considered an interference in the determination of BOD, and the inclusion of ammonia in the dilution water contributes an external source of nitrogenous demand. The interference from nitrogenous demand can now be prevented by an inhibitory chemical.
1 If an inhibiting chemical is not
used, the oxygen demand measured is the sum of carbona- ceous and nitrogenous demands.

Measurements that include nitrogenous demand generally are not useful for assessing the oxygen demand associated with organic material. Nitrogenous demand can be estimated directly from am- monia nitrogen (Section 4500-NH
3); and carbonaceous demand can
be estimated by subtracting the theoretical equivalent of the nitrite and nitrate produced in uninhibited test results. However, this method is cumbersome and is subject to considerable error. Chem- ical inhibition of nitrogenous demand provides a more direct and more reliable measure of carbonaceous demand.

The extent of oxidation of nitrogenous compounds during the 5-d incubation period depends on the concentration and type of microorganisms capable of carrying out this oxidation.

Such organisms usually are not present in raw or settled primary sewage in sufﬁcient numbers to oxidize sufﬁcient quantities of reduced nitrogen forms in the 5-d BOD test.

Many biological treatment plant efﬂuents contain sufﬁcient numbers of nitrifying organisms to cause nitriﬁcation in BOD tests. Because oxidation of nitrogenous compounds can occur in such samples, inhibition of nitriﬁcation as directed in 5210B.5e ) is recommended for samples of secondary efﬂuent, for samples seeded with secondary efﬂuent, and for samples of polluted waters.

3. Reference

1. Y OUNG, J.C. 1973. Chemical methods for nitriﬁcation control. J.

Water Pollut. Control Fed. 45:637.
5210 B. 5-Day BOD Test

1. General Discussion
The method consists of ﬁlling with diluted and seeded sample, to overﬂowing, an airtight bottle of speciﬁed size and incubating it at the speciﬁed temperature for 5 d. Dissolved oxygen is measured initially and after incubation, and the BOD is com- puted from the difference between initial and ﬁnal DO. Because the initial DO is determined shortly after the dilution is made, all oxygen uptake occurring after this measurement is included in the BOD measurement.
* Approved by Standard Methods Committee, 2001. Editorial revisions, 2011.

Joint Task Group: 21st Edition—James C. Young (chair), George T. Bowman, Sabry M. Kamhawy, Terry G. Mills, Marlene Patillo, Ray C. Whittemore. 1 Exhibit "B"
For sampling and storage procedures, see 5210B.4 a.

2. Apparatus
a. Incubation bottles: Use glass bottles having 60 mL or greater capacity (300-mL bottles having a ground-glass stopper and a ﬂared mouth are preferred). Clean bottles with a detergent, rinse thoroughly, and drain before use.
b. Air incubator or water bath, thermostatically controlled at 20 /H110061°C. Exclude all light to prevent possibility of photosyn- thetic production of DO.

3. Reagents
Prepare reagents in advance but discard if there is any sign of precipitation or biological growth in the stock bottles. Commer- cial equivalents of these reagents are acceptable and different stock concentrations may be used if doses are adjusted propor- tionally. Use reagent grade or better for all chemicals and use distilled or equivalent water, preferably sterilized, for making all solutions.
a. Phosphate buffer solution: Dissolve 8.5 g KH
2PO4, 21.75 g
K2HPO4,3 3 . 4gN a 2HPO4 /H185287H2O, and 1.7 g NH 4Cl in about 500 mL distilled water and dilute to 1 L. The pH should be

7.2 without further adjustment. Alternatively, dissolve 42.5 g KH
2PO4 and 1.7 g NH 4Cl in about 700 mL distilled water.

Adjust pH to 7.2 with 30% NaOH and dilute to 1 L.
b. Magnesium sulfate solution: Dissolve 22.5 g MgSO 4 /H185287H2O in distilled water and dilute to 1 L.
c. Calcium chloride solution: Dissolve 27.5 g CaCl 2 in dis - tilled water and dilute to 1 L.
d. Ferric chloride solution: Dissolve 0.25 g FeCl 3 /H185286H2Oi n distilled water and dilute to 1 L.
e. Acid and alkali solutions, 1N, for neutralization of caustic or acidic waste samples.
1) Acid—Slowly and while stirring, add 28 mL conc sulfuric acid to distilled water. Dilute to 1 L.
2) Alkali—Dissolve 40 g sodium hydroxide in distilled water.

Dilute to 1 L.
f. Sodium sulﬁte solution: Dissolve 1.575 g Na 2SO3 in 1000 mL distilled water. This solution is not stable; prepare daily. g. Nitriﬁcation inhibitor:
1) 2-chloro-6-(trichloromethyl) pyridine—Use pure TCMP or commercial preparations.*
2) Allylthiourea (ATU) solution—Dissolve 2.0 g allylthio- urea (C
4H8N2S) in about 500 mL water and dilute to 1 L. Store
at 4°C. The solution is stable for not more than 2 weeks. h. Glucose-glutamic acid solution: Dry reagent-grade glucose and reagent-grade glutamic acid at 103°C for 1 h. Add 150 mg glucose and 150 mg glutamic acid to distilled water and dilute to 1 L. Prepare fresh immediately before use unless solution is maintained in a sterile condition. Store all glucose-glutamic acid mixtures at 4°C or lower. Commercial preparations may be used but concentrations may vary.
i. Ammonium chloride solution: Dissolve 1.15 g NH
4Cl in
about 500 mL distilled water, adjust pH to 7.2 with NaOH solution, and dilute to 1 L. Solution contains 0.3 mg N/mL. j. Source water for preparing BOD dilution water: Use de- mineralized, distilled, tap, or natural water for making sample dilutions (see 5210B.4 c).

4. Preparatory Procedures
a. Sampling and storage: Samples for BOD analysis may
degrade signiﬁcantly during storage between collection and anal- ysis, resulting in low BOD values.
1) Grab samples—If analysis is begun withi n2ho f collec- tion, cold storage is unnecessary. If analysis is not started within 2 h of sample collection, keep sample at or below 4°C from the time of collection. Begin analysis withi n6ho f collection; when this is not possible because the sampling site is distant from the laboratory, store at or below 4°C and report length and temper- ature of storage with the results. In no case start analysis more than 24 h after grab sample collection. When samples are to be used for regulatory purposes make every effort to deliver sam- ples for analysis withi n6ho f collection.
2) Composite samples—Keep samples at or below 4°C during compositing. Limit compositing period to 24 h. Use the same criteria as for storage of grab samples, starting the measurement of holding time from end of compositing period. State storage time and conditions as part of the results.
b. Sample preparation and pretreatment:
1) All samples—Check pH; if it is not between 6.0 and 8.0, adjust sample temperature to 20 /H110063°C, then adjust pH to 7.0 to

7.2 using a solution of sulfuric acid (H
2SO4) or sodium hydrox -
ide (NaOH) of such strength that the quantity of reagent does not dilute the sample by more than 0.5%. Exceptions may be justi- ﬁed with natural waters when the BOD is to be measured at in-situ pH values. The pH of dilution water should not be affected by the lowest sample dilution. Always seed samples that have been pH adjusted.
2) Samples containing residual chlorine compounds—If pos- sible, avoid samples containing residual chlorine by sampling ahead of chlorination processes. If residual chlorine is present, dechlorinate sample. In some samples chlorine will dissipate within 1 t o2ho f standing in the light. This dissipation often occurs during sample transport and handling. For samples in which chlorine residual does not dissipate in a reasonably short time, destroy chlorine residual by adding Na
2SO3 solution. De -
termine required volume of Na 2SO3 solution on a 100- to 1000-mL portion of neutralized sample by adding 10 mL 1 /H110011 acetic acid or 1 /H1100150 H 2SO4, 10 mL potassium iodide (KI) solution (10 g /100 mL) per 1000 mL sample and titrating with Na2SO3 solution to the starch-iodine end point for residual. Add to neutralized sample the proportional volume of Na 2SO3 solu- tion determined by the above test, mix, and after 10 to 20 min check sample for residual chlorine. (N
OTE: Excess Na2SO3 exerts
an oxygen demand and reacts slowly with certain organic chlo- ramine compounds that may be present in chlorinated samples.) Do not test chlorinated/dechlorinated samples without seeding. 3) Samples containing other toxic substances—Certain indus- trial wastes, for example, plating wastes, contain toxic metals.

Such samples often require special study and treatment. 4) Samples supersaturated with DO—Samples containing DO concentration above saturation at 20°C may be encountered in cold waters or in water where photosynthesis occurs. To prevent loss of oxygen during incubation of such samples, reduce DO to * Nitriﬁcation Inhibitor Formula 2533 (2% TCMP on sodium sulfate), Hach Co., Loveland, CO, or equivalent.

BIOCHEMICAL OXYGEN DEMAND (5210)/5-Day BOD Test
2 BIOCHEMICAL OXYGEN DEMAND (5210)/5-Day BOD Test
saturation by bringing sample to about 20 /H110063°C in partially ﬁlled bottle while agitating by vigorous shaking or by aerating with clean, ﬁltered compressed air.
5) Samples containing hydrogen peroxide—Hydrogen perox- ide remaining in samples from some industrial bleaching pro- cesses such as those used at paper mills and textile plants can cause supersaturated oxygen levels in samples collected for BOD testing. Mix such samples vigorously in open containers for sufﬁcient time to allow the hydrogen peroxide to dissipate before setting up BOD tests. Check adequacy of peroxide re- moval by observing dissolved oxygen concentrations over time during mixing or by using peroxide-speciﬁc test strips. Mixing times can vary from 1 t o 2 h depending on the amount of hydrogen peroxide present. The peroxide reaction can be con- sidered complete when the DO no longer increases during a 30-min period without mixing.
c. Selection and storage of source water for BOD sample dilution: Obtain water from suitable source— distilled, tap, or receiving water. Make sure the water is free of heavy metals, speciﬁcally copper, and toxic substances, such as chlorine, that can interfere with BOD measurements. Protect source water quality by using clean glassware, tubing, and bottles. Deionized water often contains sufﬁcient amounts of organics and micro- organisms to cause failure of the dilution water quality control check (5210B.6 c). Source water may be stored before use as long as the prepared dilution water (5210B.5 a) meets quality control criteria in the dilution water blank (5210B.6 c). Such storage may improve the quality of some source waters but may allow biological growth to cause deterioration in others. Storage of prepared dilution water (5210B.5 h) for more than 24 h after adding nutrients, minerals, and buffer is not recommended un- less dilution water blanks consistently meet quality control lim- its. Discard stored source water if the dilution water blank shows more than 0.20 mg/L DO depletion i n 5 d (5210B.6 c).
d. Preparation of seed suspension: It is necessary to have present in each BOD bottle a population of microorganisms capable of oxidizing the biodegradable organic matter in the sample. Domestic wastewater, unchlorinated or otherwise undis- infected efﬂuents from biological wastewater treatment plants, and surface waters receiving wastewater discharges usually con- tain satisfactory microbial populations. Some samples (for ex- ample, some untreated industrial wastes, disinfected wastes, high-temperature wastes, wastes having pH values less than 6 or greater than 8, or wastes stored more tha n 6 h after collection) do not contain a sufﬁcient microbial population. Seed such samples by adding a population of suitable microorganisms. The pre- ferred seed is obtained from a biological treatment system pro- cessing the waste. In this case, use supernatant from settled domestic wastewater, efﬂuent from primary clariﬁers, diluted mixed liquor from an aeration basin, undisinfected efﬂuent, or receiving water from below the point of discharge. When efﬂu- ent or mixed liquor from a biological treatment process is used as a seed source, inhibition of nitriﬁcation is recommended. Do not use seed from efﬂuents that have been disinfected by chlo- rine or other means. Commercial seed sources may be used but are more likely to be unadapted to the wastewater constituents.

Do not ﬁlter seed sources; ﬁltering removes the seed microor- ganisms.

When acclimated seed sources are not available, develop an acclimated seed in the laboratory by continuously aerating a sample of settled domestic wastewater and adding small daily increments of sample from the waste in question. Use a soil suspension, activated sludge, or a commercial seed preparation to obtain the initial microbial population. Determine the exis- tence of a satisfactory population by testing the performance of the seed in BOD tests on the sample. BOD values that increase with time of adaptation to a steady high value indicate successful seed acclimation.

5. Testing Procedure
a. Preparation of dilution water: Transfer desired working volume of source water (5210B.4 c) to a suitably sized bottle (glass is preferred). Check to ensure that the dissolved oxygen concentration is at least 7.5 mg/L before using water for BOD tests. If not, add DO by shaking bottle or by aerating with organic-free ﬁltered air. Alternatively, store the water in cotton- plugged bottles long enough for the DO concentration to ap- proach saturation. Add 1 mL each of phosphate buffer, MgSO 4,
CaCl2, and FeCl 3 solution/L to prepared source water
(5210B.4c). Mix thoroughly and bring temperature to 20 /H110063°C.

Prepare dilution water immediately before use unless dilution water blanks (5210B.6 c) show that the water is acceptable after longer storage times. If the dilution water blanks show a DO depletion greater than 0.20 mg/L, obtain a satisfactory water by improving puriﬁcation or use water from another source. Do not add oxidizing agents or expose dilution water to ultraviolet light in attempts to bring the dilution blank into range.
b. Sample temperature adjustment: Bring samples to 20 /H11006 3°C before making dilutions.
c. Preparation of dilutions: Using the dilution water prepared as in ¶ a above, make at least three dilutions of prepared sample estimated to produce a residual DO of at least 1.0 mg/L and a DO uptake of at least 2.0 mg/L after a 5-d incubation. Five dilutions are recommended if experience with a particular sample does not produce at least three bottles having acceptable minimum DO depletions and residual limits (5210B.6a). A more rapid analysis, such as COD (Section 5220), may be correlated approximately with BOD and serve as a guide in selecting dilutions. In the absence of prior knowledge, use the following percentages of wastewater when preparing dilutions: 0.01 to 1.0% for strong industrial wastes, 1 to 5% for raw and settled wastewater, 5 to 25% for biologically treated efﬂuent, and 25 to 100% for pol- luted river waters. The number of bottles to be prepared for each dilution depends on the DO technique and the number of repli- cates desired. Prepare dilutions in volumetric containers (Class A glass or equivalent) and then transfer to BOD bottles or prepare directly in BOD bottles. Either dilution method can be combined with any DO measurement technique.
1) Dilutions prepared in volumetric containers—Using a
wide-tipped pipet, add desired amount of prepared sample to individual volumetric cylinders or ﬂasks. Mix the sample well immediately before pipetting to avoid loss of solids by settling.

For dilutions greater than 1:100 make a primary dilution before making ﬁnal dilution in the bottle. Fill cylinders or ﬂasks at least two-thirds full of dilution water without entraining air. Add appropriate amounts of seed suspension (¶ d below) and nitriﬁ- cation inhibitor (¶ e below). Dilute to ﬁnal level with dilution water (¶ a above). Mix well but avoid entraining air. Siphon BIOCHEMICAL OXYGEN DEMAND (5210)/5-Day BOD Test
3 BIOCHEMICAL OXYGEN DEMAND (5210)/5-Day BOD Test
mixed dilution into a suitable number of BOD bottles, taking care not to let solids settle in the cylinder or ﬂask during transfer. 2) Dilutions prepared directly in BOD bottles—Using a wide- tip volumetric pipet, add the desired sample volume to individual BOD bottles. Fill each BOD bottle approximately two-thirds full with dilution water. Add appropriate amounts of seed suspension (¶ d below) and nitriﬁcation inhibitor (¶ e below) to the individ- ual BOD bottles. When a bottle contains more than 67% of the sample after dilution, nutrients may be limited in the diluted sample and subsequently reduce biological activity. In such samples, add the nutrient, mineral, and buffer solutions (5210B.3a–e) directly to diluted sample at a rate of 1 mL/L (0.30 mL/300-mL bottle) or use commercially prepared solu- tions designed to dose the appropriate bottle size.
d. Addition of seed suspension: If seeding is used, add seed suspensions to the dilution vessels or to individual BOD bottles before ﬁnal dilution as described in ¶ c above. Do not add seed directly to wastewater samples if they contain materials that are toxic before dilution. Generally, 1 to 3 mL of settled raw waste- water or primary efﬂuent or 1 to 2 mL of a 1:10 dilution of mixed liquor/300-mL bottle will provide a suitable amount of micro- organisms. Do not ﬁlter seed suspension before use. Agitate the seed suspension during transfer to ensure that the same quantity of microorganisms is added to each BOD bottle. Always record the exact volume of seed suspension added to each bottle. The DO uptake attributable to the seed added to each bottle generally should be between 0.6 and 1.0 mg/L, but the amount of seed added should be adjusted from this range to that required to provide glucose-glutamic acid (GGA) check results of 198 /H11006

30.5 mg/L. For example, if 1 mL of seed suspension is required to achieve 198 /H1100630.5 mg/L BOD in the glucose-glutamic acid check, then use 1 mL in each BOD bottle receiving the test wastewater.
e. Addition of nitriﬁcation inhibitor: Samples that may require nitriﬁcation inhibition
1 include, but are not limited to, biologi -
cally treated efﬂuents, samples seeded with biologically treated efﬂuents, and river waters. Note the use of nitrogen inhibition and the chemical used when reporting results. (N
OTE: TCMP is
the preferred nitriﬁcation inhibitor but requires handling and transfer in a solid form. Allylthiourea is not always effective in inhibiting nitriﬁcation within the 5-d incubation period and con- centrations above 2 mg/L may cause increases in carbonaceous BOD measurements. ATU concentrations above 2 mg/L also can adversely affect the azide modiﬁcation of the iodometric method). Seed all samples to which nitriﬁcation inhibitor has been added. The amount of seed should be consistent with that required to achieve GGA test results in the range of 198 /H1100630.5 mg/L (5210B.6 b).
1) Nitriﬁcation inhibition using 2-chloro-6-(trichloromethyl) pyridine (TCMP)—Add 10 mg TCMP/L to diluted sample or
3 mg TCMP to each 300-mL bottle or sample dilution vessel, or proportional amounts to other sized bottles, after initial sample dilution but before ﬁnal ﬁlling of the bottles with dilution water.

Do not add TCMP to BOD bottles before they are at least two-thirds ﬁlled with diluted sample. (N
OTE: TCMP dissolves
slowly and can ﬂoat on top of the sample if not mixed well).

Some commercial TCMP formulations are not 100% TCMP;
adjust dosage appropriately.
2) Nitriﬁcation inhibition using allylthiourea (ATU)—Add 1 mL ATU solution (5210B.3 g2)/L diluted sample or 0.3 mL/ 300mL test bottle or sample dilution vessel. Do not add ATU to BOD bottles until they are at least two-thirds ﬁlled with diluted sample.
f. Sealing of bottles: Complete ﬁlling of each bottle by adding enough dilution water that insertion of the stopper leaves no bubbles in the bottle. Mix the sample by turning the bottle manually several times unless a DO probe having a stirrer is used immediately to measure initial DO concentration. As a precau- tion against drawing air into the dilution bottle during incuba- tion, use a water seal. Obtain satisfactory water seals by invert- ing bottles in a water bath or by adding water to the ﬂared mouth of special BOD bottles. Place a paper or plastic cup or foil cap over ﬂared mouth of bottle to reduce evaporation of the water seal during incubation.
g. Determination of initial DO: Use the azide modiﬁcation of the iodometric method (Section 4500-O.C) or the membrane electrode method (Section 4500-O.G) to determine initial DO on all sample dilutions, dilution water blanks, and, where appropri- ate, seed controls. Replace any displaced contents with sufﬁcient diluted sample or dilution water to ﬁll the bottle, stopper all bottles tightly, and water seal before beginning incubation. After preparing dilution, measure initial DO within 30 min. If the membrane electrode method is used, take care to eliminate drift in calibration between initial and ﬁnal DO readings. If the azide modiﬁcation of the titrimetric iodometric method is used, pre- pare an extra bottle for initial DO determination for each sample dilution.
h. Sample incubation: Incubate at 20°C /H110061°C the stoppered and sealed BOD bottles containing desired dilutions (¶ a above), seed controls (5210B,6 d), dilution water blanks (5210B.6 c), and glucose-glutamic acid checks (5210B.6 b). Exclude light to avoid growth of algae in the bottles during incubation.
i. Determination of ﬁnal DO: After 5 d /H110066 h of incubation, determine DO in all sample dilutions, and in all blanks and checks as in ¶ 6 b– d, using the azide modiﬁcation of the titrim- etric method or the membrane electrode method.

6. Quality Control Checks
The quality control practices considered to be an integral part of each method are summarized in Table 5020:I.
a. Minimum residual DO and minimum DO depletion: Only
bottles, including seed controls, giving a minimum DO depletion of 2.0 mg/L and a residual DO of at least 1.0 mg/L afte r5do f incubation are considered to produce valid data, because at least

2.0 mg oxygen/uptake L is required to give a meaningful mea- sure of oxygen uptake and at least 1.0 mg/L must remain throughout the test to ensure that insufﬁcient DO does not affect the rate of oxidation of waste constituents. Exceptions occur for reporting purposes only when the depletions for tests using undiluted samples in all bottles fall below 2.0 mg/L and when the residual DO in all dilutions is less than 1.0 mg/L (5210B.7).

When using membrane electrodes for measuring DO, make
frequent calibration checks to ensure accurate DO readings (Sec- tion 4500-0.G.3 a).
b. Glucose-glutamic acid check: The glucose-glutamic acid check is the primary basis for establishing accuracy and preci- sion of the BOD test and is the principal measure of seed quality and set-up procedure. Together with each batch of samples, check seed effectiveness and analytical technique by using pro- BIOCHEMICAL OXYGEN DEMAND (5210)/5-Day BOD Test
4 BIOCHEMICAL OXYGEN DEMAND (5210)/5-Day BOD Test
cedures in 5210B.5 to make BOD measurements on an equal weight mixture of glucose and glutamic acid as follows: Add sufﬁcient amounts of standard glucose-glutamic acid solution (5210B.3h) to give 3.0 mg glucose/L and 3.0 mg glutamic acid/L in each of three test bottles (20 mL GGA solution/L seeded dilution water, or 6.0 mL/300-mL bottle). Commercial solutions may contain other glucose-glutamic acid concentra- tions; adjust doses accordingly. Add nitriﬁcation inhibitor if seed is obtained from a source that is nitrifying. Evaluate data as described in 5210B.8, Precision and Bias. The resulting average BOD for the three bottles, after correction for dilution and seeding, must fall into the range of 198 /H1100630.5 mg/L. If the average value falls outside this range, evaluate the cause and make appropriate corrections. Consistently high values can in- dicate the use of too much seed suspension, contaminated dilu- tion water, or the occurrence of nitriﬁcation; consistently low values can indicate poor seed quality or quantity or the presence of a toxic material. If low values persist, prepare a new mixture of glucose and glutamic acid and check the sources of dilution water and source of seed.
c. Dilution water quality check: With each batch of samples incubate one or more bottles of dilution water that contains nutrient, mineral, and buffer solutions but no seed or nitriﬁcation inhibitor. This dilution water blank serves as a check on quality of unseeded dilution water and cleanliness of incubation bottles.

Determine initial and ﬁnal DO as in 5210B.5 g and i. The DO uptake i n 5 d must not be more than 0.20 mg/L and preferably not more than 0.10 mg/L, before making seed corrections. If the dilution water blank exceeds 0.20 mg/L, discard all data for tests using this dilution water or clearly identify such samples in data records.
d. Seed control: Determine BOD of the seed suspension as for any other sample. This is the seed control. Ideally, make three dilutions of seed such that the smallest quantity gives at least

2.0 mg/L DO depletion and the largest quantity results in at least

1.0 mg/L DO residual after 5 d of incubation. Determine the DO uptake per milliliter of seed added to each bottle using either the slope method or the ratio method. For the slope method, plot DO depletion in milligrams per liter versus milliliters of seed for all seed control bottles having a 2.0 mg/L depletion and 1.0 mini- mum residual DO. The plot should present a straight line for which the slope indicates DO depletion per milliliter of seed. The DO-axis intercept is oxygen depletion caused by the dilution water and should be less than 0.20 mg/L (see ¶ c above). For the ratio method, divide the DO depletion by the volume of seed in milliliters for each seed control bottle having a 2.0 mg/L deple- tion and greater than 1.0 mg/L minimum residual DO and
average the results. Seed dilutions showing widely varying de- pletions per milliliter of seed ( /H1100630%) suggest the presence of toxic substances or large particulates in the seed suspension. In this case, check or change the seed source.

7. Data Analysis and Reporting
a. Calculations:
1) For each test bottle having 2.0 mg/L minimum DO deple- tion and at least 1.0 mg/L residual DO, calculate BOD as follows:

BOD5, mg/L /H11005(D1 /H11002D2) /H11002(S)Vs
P
where:

D1 /H11005DO of diluted sample immediately after preparation, mg/L, D2 /H11005DO of diluted sample afte r 5 d incubation at 20°C, mg/L, S /H11005oxygen uptake of seed, /H9004DO/mL seed suspension added per bottle (5210B.6 d)( S /H110050 if samples are not seeded), Vs /H11005volume of seed in the respective test bottle, mL, and P /H11005decimal volumetric fraction of sample used; 1/ P /H11005dilution factor.
2) If DO depletion is less than 2.0 mg/L and sample concen- tration is 100% (no dilution except for seed, nutrient, mineral, and buffer solutions), actual seed-corrected, DO depletion may be reported as the BOD even if it is less than 2.0 mg/L. 3) When all dilutions result in a residual DO /H110211.0, select the bottle having the lowest DO concentration (greatest dilution) and report:

BOD, mg/L /H11022(D1 /H11002D2) /H11002(S)Vs
P
In the above calculations, do not make corrections for DO uptake by the dilution water blank during incubation. This cor- rection is unnecessary if dilution water meets the blank criteria stipulated in 5210B.6 c. If the dilution water does not meet these criteria, proper corrections are difﬁcult; do not record results or, as a minimum, mark them as not meeting quality control criteria. b. Reporting: Average the test results for all qualiﬁed bottles within each dilution series. Report the result as BOD
5 if nitriﬁ -
cation is not inhibited. Report results as CBOD 5 if nitriﬁcation is inhibited. Samples showing large differences between the com- puted BOD for different dilutions, for example, greater than 30%, may indicate the presence of a toxic substance or analytical problems. When the effect becomes repetitive, investigate to identify the cause. Identify results in the test reports when any of the following quality control parameters is not met:
• Dilution water blank exceeds 0.20 mg/L (5210B.6 c),
• Glucose-glutamic acid check falls outside acceptable limits (5210B.6b),
• Test replicates show more than 30% difference between high and low values,
• Seed control samples do not meet the above criteria in all dilutions (5210B.6 d), or
• Minimum DO is less than 1.0 mg/L [5210B.7 a3)].

8. Precision and Bias
There is no measurement for establishing bias of the BOD procedure. The glucose-glutamic acid check prescribed in 5210B.6b is intended to be a reference point for evaluation of dilution water quality, seed effectiveness, and analytical tech- nique. Single-laboratory tests using a 300-mg/L mixed glucose- glutamic acid solution provided the following results:

Number of months: 14
Number of triplicates: 421
Average monthly recovery: 204 mg/L
Average monthly standard deviation: 10.4 mg/L
BIOCHEMICAL OXYGEN DEMAND (5210)/5-Day BOD Test
5 BIOCHEMICAL OXYGEN DEMAND (5210)/5-Day BOD Test
In a series of interlaboratory studies, 2 each involving 2 to 112 laboratories (and as many analysts and seed sources), 5-d BOD measurements were made on synthetic water samples containing a 1:1 mixture of glucose and glutamic acid in the total concen- tration range of 3.3 to 231 mg/L. The regression equations for mean value, X, and standard deviation, S, from these studies were:

X /H110050.658 (added concentration, mg/L) /H110010.280 mg/L S /H110050.100 (added concentration, mg/L) /H110010.547 mg/L For the 300-mg/L mixed primary standard, the average 5-d BOD would be 198 mg/L with a standard deviation of

30.5 mg/L. When nitriﬁcation inhibitors are used, GGA test results falling outside the 198 /H1100630.5 control limit quite often indicate use of incorrect amounts of seed. Adjust amount of seed added to the GGA test to achieve results falling within this range. a. Control limits: Because of many factors affecting BOD tests in multilaboratory studies and the resulting extreme variability in test results, one standard deviation, as determined by interlabo- ratory tests, is recommended as a control limit for individual laboratories. Alternatively, each laboratory may establish its control limits by performing a minimum of 25 glucose-glutamic acid checks (5210B.6b) over a period of several weeks or months and calculating the mean and standard deviation. Use the mean /H110063 standard deviations as the control limit for future glucose- glutamic acid checks. Compare calculated control limits to the single-laboratory tests presented above and to interlaboratory results. If the glucose-glutamic acid test results are outside the range of 198 /H1100630.5, re-evaluate the control limits and investi- gate source of the problem. If measured BOD for a glucose- glutamic acid check is outside the accepted control limit range, reject tests made with that seed and dilution water or identify such tests clearly in all data records and reports.
b. Working range and detection limit: The working range is equal to the difference between the maximum initial DO (7 to 9 mg/L) and minimum DO residual of 1 mg/L corrected for seed, and multiplied by the dilution factor.

Detection limits are established by the minimum DO depletion and minimum DO residuals as follows:
• The lower detection limit for unseeded samples that require dilution ( S /H110050; P /H110211.0) is 2 mg/L multiplied by the dilution factor as established by the requirement for a minimum DO depletion of 2 mg/L.
• The lower limit for seeded samples that require dilution (S /H110220; P /H110211.0) is approximately 1 mg/L as established by the minimum depletion of 2.0 mg/L minus the maximum seed cor- rection, which should be less than about 1 mg/L.
• The lower limit for unseeded samples that require no dilution (S /H110050; P /H110051.0) is equal to the detection limit of the DO measurement method ( /H110110.1 mg/L).
• The lower detection limit for seeded samples that require no dilution ( S /H110220; P /H110051.0) is 0 mg/L, as established by the difference between the sample DO depletion and the seed cor- rection.

9. References

1. Y OUNG, J.C. 1973. Chemical methods for nitriﬁcation control. J.

Water Pollut. Control Fed. 45:637.

2. U.S. E NVIRONMENTAL PROTECTION AGENCY,O FFICE OF RESEARCH AND DEVELOPMENT. 1986. Method-by-Method Statistics from Water Pol- lution (WP) Laboratory Performance Evaluation Studies. Quality Assurance Branch, Environmental Monitoring and Support Lab., Cincinnati, Ohio.

10. Bibliography
THERIAULT, E.J., P.D. M CNAMEE & C.T. B UTTERFIELD. 1931. Selection of dilution water for use in oxygen demand tests. Pub. Health Rep. 46:1084.

LEA, W.L. & M.S. N ICHOLS. 1937. Inﬂuence of phosphorus and nitrogen on biochemical oxygen demand. Sewage Works J. 9:34.

RUCHHOFT, C.C. 1941. Report on the cooperative study of dilution waters made for the Standard Methods Committee of the Federation of Sewage Works Associations. Sewage Works J. 13:669.

M
OHLMAN, F.W., E. H URWITZ, G.R. B ARNETT & H.K. R AMER. 1950.

Experience with modiﬁed methods for BOD. Sewage Ind. Wastes 22:31.

YOUNG, J.C., G.N. M CDERMOTT &D .J ENKINS . 1981. Alterations in the BOD procedure for the 15th edition of Standard Methods for the Examination of Water and Wastewater. J. Water Pollut. Control Fed. 53:1253.
5210 C. Ultimate BOD Test

1. General Discussion
The ultimate BOD test is an extension of the 5-d dilution BOD test (5210B) but with a number of speciﬁc test requirements and differences in application. The user should be familar with the 5210B procedure before conducting tests for UBOD.
a. Principle: The method consists of placing a single sample dilution in full, airtight bottles and incubating under speciﬁed con- ditions for an extended period depending on wastewater, efﬂuent, river, or estuary quality.
1 Dissolved oxygen (DO) is measured (with
probes) initially and intermittently during the test. From the DO versus time series, UBOD is calculated by an appropriate statistical technique. For improved accuracy, run tests in triplicate.

Bottle size and incubation time are ﬂexible to accommodate individual sample characteristics and laboratory limitations. In- cubation temperature, however, is 20°C. Most efﬂuents and some naturally occurring surface waters contain materials with oxygen demands exceeding the DO available in air-saturated water. Therefore, it is necessary either to dilute the sample or to monitor DO frequently to ensure that low DO or anaerobic conditions do not occur. When DO concentrations approach 2 mg/L, the sample should be reaerated.

Because bacterial growth requires nutrients such as nitrogen, phosphorus, and trace metals, the necessary amounts may be added to the dilution water together with buffer to ensure that pH remains in a range suitable for bacterial growth and seed to BIOCHEMICAL OXYGEN DEMAND (5210)/Ultimate BOD Test
6 BIOCHEMICAL OXYGEN DEMAND (5210)/Ultimate BOD Test
provide an adequate bacterial population. However, if the result is being used to estimate the rate of oxidation of naturally occurring surface waters, addition of nutrients and seed probably accelerates the decay rate and produces misleading results. If only UBOD is desired, it may be advantageous to add supple- mental nutrients that accelerate decay and reduce the test dura- tion. When nutrients are used, they also should be used in the dilution water blank. Because of the wide range of water and wastewater characteristics and varied applications of UBOD data, no speciﬁc nutrient or buffer formulations are included.

The extent of oxidation of nitrogenous compounds during the prescribed incubation period depends on the presence of micro- organisms capable of carrying out this oxidation. Such organ- isms may not be present in wastewaters in sufﬁcient numbers to oxidize signiﬁcant quantities of reduced nitrogen. This situation may be reversed in naturally occurring surface waters. Erratic results may be obtained when a nitriﬁcation inhibitor is used; 2 therefore, the speciﬁed method precludes use of a nitrogen inhibitor unless prior experimental evidence on the particular sample suggests that it is acceptable.* Monitor NO
2 /H11002-N and
NO3
/H11002-N to compute the oxygen equivalency of the nitriﬁcation reaction. When these values are subtracted from the DO vs. time series, the carbonaceous BOD time series can be constructed. 3 b. Sampling and storage: See 5210B.4 a.
c. Quality control (QC): The QC practices considered to be an integral part of each method are summarized in Table 5020:I.

2. Apparatus
a. Incubation bottles: Glass bottles with ground-glass stop- pers,† 2-L (or larger) capacity. Glass serum bottles of 4- to 10-L capacity are available. Alternatively use nonground-glass bottles with nonbiodegradable plastic caps as a plug insert. Do not reuse the plugs because discoloration occurs with continued use. Re- place plugs every 7 to 14 d. Do not use rubber stoppers that may exert an oxygen demand. Clean bottles with a detergent and wash with dilute HCl (3 N) to remove surface ﬁlms and precip- itated inorganic salts; rinse thoroughly with DI water before use.

Cover top of bottles with paper after rinsing to prevent dust from collecting. To prevent drawing air into the sample bottle during incubation, use a water seal. If the bottle does not have a ﬂared mouth, construct a water seal by making a watertight dam around the stopper (or plug) and ﬁll with water from the reservoir as necessary. Cover dam with clean aluminum foil to retard evap- oration. If a 2-L BOD bottle is used, ﬁll reservoir with sample and cover with a polyethylene cap before incubation.

Place a clean magnetic stirring bar in each bottle to mix contents before making DO measurement or taking a subsample.

Do not remove the magnets until the test is complete.

Alternatively, use a series of 300-mL BOD bottles (5210B.2 a) if larger bottles are not available or incubation space is limited. b. Reservoir bottle: 4-L or larger glass bottle. Close with screw plastic cap or non-rubber plug.
c. Incubator or water bath , thermostatically controlled at 20 /H11006 1°C. Exclude all light to prevent the possibility of photosynthetic production of DO.
d. Oxygen-sensitive membrane electrode: See Section 4500- O.G.2.

3. Procedure
a. River water samples: Preferably ﬁll large BOD bottle (/H110222 L, or alternatively 6 or more 300-mL BOD bottles) with sample at 20°C. Add no nutrients, seed, or nitriﬁcation inhibitor if in-bottle decay rates will be used to estimate in-stream rates.

Do not dilute sample unless it is known by pretesting or by experience to have a high ultimate BOD ( /H1102220 mg/L).

Measure DO in each bottle, stopper, and make an airtight seal.

Incubate at 20°C in the dark.

Measure DO in each bottle at intervals of at least 2 t o 5 d over a period of 30 to 60 d (minimum of 6 to 8 readings) or longer under special circumstances. To avoid oxygen depletion in sam- ples containing NH
3-N, measure DO more frequently until ni -
triﬁcation has taken place. If DO falls to about 2 mg/L, reaerate as directed below. Replace sample lost by the cap and DO probe displacement by adding 1 to 2 mL sample from the reservoir bottle.

When DO approaches 2 mg/L, reaerate. Pour a small amount of sample into a clean vessel and reaerate the remainder directly in the bottle by vigorous shaking or bubbling with puriﬁed air (medical grade). Reﬁll bottle from the storage reservoir and measure DO. This concentration becomes the initial DO for the next measurement. If using 300-mL BOD bottles, pour all of the sample from the several bottles used into a clean vessel, reaerate, and reﬁll the small bottles.

Analyze for nitrate plus nitrite nitrogen (NO
3 /H11002-N /H11001NO2
/H11002-N)
(see Sections 4500-NO 2
/H11002and 4500-NO3
/H11002) on Days 0, 5, 10, 15,
20, and 30. Alternatively, determine NO 2
/H11002-N and NO 3
/H11002-N each
time DO is determined, thereby producing corresponding BOD and nitrogen determinations. If the ultimate demand occurs at a time greater than 30 d, make additional analyses at 30-d inter- vals. Remove 10 to 20 mL from the bottle for these analyses.

Reﬁll bottle as necessary from the reservoir bottle. Preserve NO
2 /H11002-N /H11001NO3
/H11002-N subsample with H 2SO4 to pH /H110212 and
refrigerate. If the purpose of the UBOD test is to assess the UBOD and not to provide data for rate calculations, measure nitrate nitrogen concentration only at Day 0 and on the last day of the test (kinetic rate estimates are not useful when the nitri- ﬁcation reaction is not followed).

Calculate oxygen consumption during each time interval and make appropriate corrections for nitrogenous oxygen demand.

Correct by using 3.43 /H11003the NH
3-N to NO 2
/H11002-N conversion plus

1.14 /H11003the NO 2
/H11002-N to NO 3
/H11002-N conversion to reﬂect the stoi -
chiometry of the oxidation of NH 4
/H11001to NO 2
/H11002or NO 3
/H11002.

When using a dilution water blank, subtract DO uptake of the blank from the total DO consumed. High-quality reagent water without nutrients typically will consume a maximum of 1 mg DO/L in a 30- to 90-d period. If DO uptake of the dilution water is greater than 0.5 mg/L for a 20-d period, or 1 mg/L for a 90-d period, report the magnitude of the correction and try to obtain higher-quality dilution water for use with subsequent UBOD tests.
* Some analysts have reported satisfactory results with 2-chloro-6-(trichloro- methyl) pyridine (Nitriﬁcation Inhibitor, Formula 2533, Hach Co., Loveland, CO, or equivalent).
† Wheaton 2-L BOD bottle No. 227580, 1000 North Tenth St., Millville, NJ, or equivalent.

BIOCHEMICAL OXYGEN DEMAND (5210)/Ultimate BOD Test
7 BIOCHEMICAL OXYGEN DEMAND (5210)/Ultimate BOD Test
When the weekly DO consumption drops below 1 to 2% of the total accumulative consumption, calculate the ultimate BOD using a nonlinear regression method.
b. Wastewater treatment plant samples: Use high-quality re- agent water (see Section 1080) for dilution water. Add no nitri- ﬁcation inhibitors if decay rates are desired. If seed and nutrients are necessary, add the same amounts of each to the dilution water blank. Use minimal sample dilution. As a rule of thumb, the ultimate BOD of the diluted sample should be in the range of 20 to 30 mg/L. Dilution to this level probably will require two or three sample reaerations during the incubation period to avoid having dissolved oxygen concentrations fall below 2 mg/L.

Use 2-L or larger BOD bottles (alternatively, multiple 300-mL BOD bottles) for each dilution. Add desired volume of sample to each bottle and ﬁll with dilution water.

Fill a BOD bottle with dilution water to serve as a dilution water blank. Treat blank the same as all samples. Follow procedure given in ¶ a above and incubate for at least as long as UBOD test.

4. Calculations
An example of results obtained for a wastewater sample, undiluted, without seed and nutrients, is given in Table 5210:I.

UBOD can be estimated by using a ﬁrst-order model described as follows:

BOD t /H11005UBOD (1 /H11002e/H11002kt)
where:

BODt /H11005oxygen uptake measured at time t, mg/L, and k /H11005ﬁrst-order oxygen uptake rate.

The data in Table 5210:I were analyzed with a nonlinear regression technique applied to the above ﬁrst-order model. 4 However, a ﬁrst-order kinetic model may not always be the best choice. Signiﬁcantly better statistical ﬁts usually are obtained with alternative kinetic models including sum of two ﬁrst-order and logistic function models.
1,3– 8

5. Precision and Bias
The precision of the ultimate BOD test was assessed with a series of replicate tests in a single laboratory. Interlaboratory studies have not been conducted.

Reference Replicate No.

UBOD
mg/L Precision Summary*
2 1 154 /H9262/H11005151 mg/L
2 154
3 145 CV /H110053.5%
5 1 10.3
2 11.1
3 9.6
/H9262/H1100510.0 mg/L
4 9.9
5 9.8 CV /H110055.8%
6 9.6
6 1 12.8
/H9262/H1100512.4 mg/L
2 12.6
3 12.6 CV /H110054.4%
4 11.6
* /H9262/H11005mean,
CV /H11005coefﬁcient of variation.

TABLE 5210:I. UBOD R ESULTS FOR WASTEWATER SAMPLE
Day
(1) (2) (3) (4) (5) (6)
Average
DO*
mg/L
Average
Blank DO†
mg/L
Accumulated DO
Consumed by Sample‡
mg/L
Average
NO3-N
mg/L
NBOD
mg/L§
CBOD
mg/L/H14067
0 8.1 — 0 0.0 0 0
3 5.6 — 2.5 — 0 2.5
5 3.5/8.0 — 4.6 0.0 0 4.6
7 6.2 — 6.4 — 0.23 6.2
10 3.2/8.2 — 9.4 0.10 0.46 8.9
15 4.3 — 13.3 — 0.58 12.7
18 2.7/8.1 — 14.9 0.15 0.69 14.2
20 6.6 — 16.4 — 0.80 15.6
25 5.4 — 17.6 0.20 0.92 16.7
30 2.6/8.2 — 20.4 — 0.92 19.5
40 5.3 — 23.3 0.20 0.92 22.4
50 3.1/8.0 — 25.5 — 0.92 24.6
60 4.5 — 29.0 — 0.92 28.1
70 3.3/8.1 — 30.2 — 0.92 29.3
90 5.4 — 32.9 0.20 0.92 32.0
* Two readings indicate concentrations before and after reaeration. † None was used.
‡ Column (1) – blank correction (none needed in the example). § Column (4) /H110034.57 (linear interpolation between values). /H14067[Column (3) – Column (5)] /H11003dilution factor.

Ultimate CBOD /H1100534.5 mg/L; CBOD decay rate /H110050.03/d (calculated with ﬁrst-order equation from 5210C.4).

BIOCHEMICAL OXYGEN DEMAND (5210)/Ultimate BOD Test
8 BIOCHEMICAL OXYGEN DEMAND (5210)/Ultimate BOD Test
Bias was assessed by determining the BOD of a known
concentration of glucose (150 mg/L) and glutamic acid
(150 mg/L). This solution has a UBOD of 321 mg/L to
308 mg/L, depending on extent of nitriﬁcation. The results of the study conducted in triplicate were:
1 Estimated*
UBOD
mg/L
Theoretical
BOD
mg/L
Percent
Difference
276 308/321 /H1100210//H1100214
310 308/321 /H110011//H110023
303 308/321 /H110022//H110026
* By statistical model.

6. References

1. M ARTONE, C.H. 1976. Studies Related to the Determination of Bio- degradability and Long Term BOD. M.S. thesis, Dep. Civil Engi- neering, Tufts Univ., Medford, Mass.

2. N

ATIONAL COUNCIL OF THE PAPER INDUSTRY FOR AIR AND STREAM IMPROVEMENT,I NC. 1986. A Review of the Separation of Carbona- ceous and Nitrogenous BOD in Long-Term BOD Measurements.

Tech. Bull. No. 461, New York, N.Y.

3. N

ATIONAL COUNCIL OF THE PAPER INDUSTRY FOR AIR AND STREAM IMPROVEMENT,I NC. 1982. A Review of Ultimate BOD and Its Kinetic Formulation for Pulp and Paper Mill Efﬂuents. Tech. Bull. No. 382, New York, N.Y.

4. B
ARNWELL, T. 1980. Least Squares Estimates of BOD Parameters. J.

Environ. Eng. Div., Proc. Amer. Soc. Civil Eng. 107(EE6):1197.

5. N ATIONAL COUNCIL OF THE PAPER INDUSTRY FOR AIR AND STREAM IMPROVEMENT,I NC. 1982. A Proposal to Examine the Effect of Mixing on Long Term BOD Test. NE82-01, New York, N.Y.

6. N ATIONAL COUNCIL OF THE PAPER INDUSTRY FOR AIR AND STREAM IMPROVEMENT,I NC. 1982. A Study of the Selection, Calibration, and Veriﬁcation of Mathematical Water Quality Models. Tech. Bull. No. 367, New York, N.Y.

7. N

ATIONAL COUNCIL OF THE PAPER INDUSTRY FOR AIR AND STREAM IMPROVEMENT,I NC. 1987. User’s Manual for Parameter Estimation for First Order Ultimate BOD Decay, BODFO. Tech. Bull. No. 529, New York, N.Y.

8. C
HU, W.S. & E.W. STRECKER. 1972. Parameter Identiﬁcation In Water System Model. Dep. Civil Engineering, Univ. Washington, Seattle. 5210 D. Respirometric Method

1. General Discussion
a. Principle: Respirometric methods provide direct measure- ment of the oxygen consumed by microorganisms from an air or oxygen-enriched environment in a closed vessel under condi- tions of constant temperature and agitation.
b. Uses: Respirometry measures oxygen uptake more or less continuously over time. Respirometric methods are useful for assessing: biodegradation of speciﬁc chemicals; treatability of organic industrial wastes; the effect of known amounts of toxic compounds on the oxygen-uptake reaction of a test wastewater or organic chemical; the concentration at which a pollutant or a wastewater measurably inhibits biological degradation; the ef- fect of various treatments such as disinfection, nutrient addition, and pH adjustment on oxidation rates; the oxygen requirement for essentially complete oxidation of biologically oxidizable matter; the need for using adapted seed in other biochemical oxygen-uptake measurements, such as the dilution BOD test; and stability of sludges.

Respirometric data typically will be used comparatively, that is, in a direct comparison between oxygen uptakes from two test samples or from a test sample and a control. Because of inherent differences among uses, among seed cultures, among applica- tions of results, and among instruments, a single procedure for respirometric tests applicable to all cases cannot be deﬁned.

Therefore, only basic recommendations and guidelines for over- all test setup and procedure are given. Follow manufacturer’s instructions for operating details for speciﬁc commercial instru- ments.
c. Types of respirometers: Four principal types of commercial respirometers are available. Manometric respirometers relate oxygen uptake to the change in pressure caused by oxygen consumption while maintaining a constant volume. Volumetric respirometers measure oxygen uptake in incremental changes in gas volume while maintaining a constant pressure at the time of reading. Electrolytic respirometers monitor the amount of oxy- gen produced by electrolysis of water to maintain a constant oxygen pressure within the reaction vessel. Direct-input respi- rometers deliver oxygen to the sample from a pure oxygen supply through metering on demand as detected by minute pressure differences. Most respirometers have been instrumented to permit data collection and processing by computer. Reaction- vessel contents are mixed by using a magnetic or mechanical stirring device or by bubbling the gaseous phase within the reaction vessel through the liquid phase. All respirometers re- move carbon dioxide produced during biological growth by suspending a concentrated adsorbent (granular or solution) within the closed reaction chamber or by recirculating the gas phase through an external scrubber.
d. Interferences: Evolution of gases other than CO
2 may
introduce errors in pressure or volume measurements; this is uncommon in the presence of dissolved oxygen. Incomplete CO 2 absorption will introduce errors if appropriate amounts and con- centrations of alkaline absorbent are not used. Temperature ﬂuctuations or inadequate mixing will introduce error. Fluctua- tions in barometric pressure can cause errors with some respi- rometers. Become familiar with the limits of the instrument to be used.
e. Minimum detectable concentration: Most commercial re- spirometers can detect oxygen demand in increments as small as

0.1 mg but test precision depends on the total amount of oxygen consumed at the time of reading, the precision of pressure or volume measurement, and the effect of temperature and baro- metric pressure changes. Upper limits of oxygen uptake rate are BIOCHEMICAL OXYGEN DEMAND (5210)/Respirometric Method
9 BIOCHEMICAL OXYGEN DEMAND (5210)/Respirometric Method determined by the ability to transfer oxygen into the solution from the gas phase, which typically is related to mixing intensity.

Transfer limits typically range from less than 10 mg O
2/L/h for
low-intensity mixing to above 100 mg O 2/L/h for high-intensity mixing.
f. Relationship to dilution BOD: Variations in waste compo- sition, substrate concentration, mixing, and oxygen concentra- tions from one wastewater source to another generally preclude use of a general relationship between oxygen uptake by respi- rometers and the 5-d, 20°C, BOD (see 5210B). Reasonably accurate correlations may be possible for a speciﬁc wastewater.

The incubation period for respirometric measurements need not be 5 d because equally valid correlations can be made between the 5-d dilution BOD and respirometric oxygen uptake at any time after 2 d.
1,2 The point of common dilution and respirometric
BOD seems to occur at about 2 t o 3 d incubation for municipal wastewaters. Correlations between respirometric measurements and 5-d BOD for industrial wastes and speciﬁc chemicals are less certain. Respirometric measurements also can provide an indication of the ultimate biochemical oxygen demand (UBOD) (see 5210C). In many cases, it is reasonable to consider that the 28- to 30-d oxygen uptake is essentially equal to the UBOD. 3 More commonly, respirometers are used as a diagnostic tool.

The continuous readout of oxygen consumption in respirometric measurements indicates lag, toxicity, or any abnormalities in the biodegradation reaction. The change in the normal shape of an oxygen-uptake curve in the ﬁrst few hours may help to identify the effect of toxic or unusual wastes entering a treatment plant in time to make operating corrections.
g. Relationship to other test methods and protocols: This method supports most of the protocols and guidelines established by the European Organization for Economic Co-operation and Development
3 (OECD) that require measurement of oxygen
uptake.
h. Sampling and storage:
1) Grab samples—If analysis is begun withi n2ho f sample collection, cold storage is unnecessary. Otherwise, keep sample at or below 4°C from the time of collection. Begin analysis within6ho f collection; when this is not possible, store at or below 4°C and report length and temperature of storage. Never start analysis more than 24 h after grab sample collection. 2) Composite samples—Keep samples at or below 4°C during compositing. Limit compositing period to 24 h. Use the same criteria as for storage of grab samples, starting the measurement of holding time from the end of the compositing period. State storage time and conditions with results.

2. Apparatus
a. Respirometer system: Use commercial apparatus and check manufacturer’s instructions for speciﬁc system requirements, reaction vessel type and volume, and instrument operating char- acteristics.
b. Incubator or water bath: Use a constant-temperature room, incubator chamber, or water bath to control temperature to /H110061°C. Exclude all light to prevent oxygen formation by algae in the sample. Use red, actinic-coated bottles for analysis outside of a darkened incubator.

3. Reagents
Formulations of reagent solutions are given for 1-L volumes, but smaller or larger volumes may be prepared according to need. Discard any reagent showing signs of biological growth or chemical precipitation. Stock solutions can be sterilized by au- toclaving to provide longer shelf life.
a. Distilled water: Use only high-quality water distilled from a block tin or all-glass still (see Section 1080). Deionized water may be used but often contains high bacterial counts. The water must contain less than 0.01 mg heavy metals/L and be free of chlorine, chloramines, caustic alkalinity, organic material, or acids. Make all reagents with this water. When other waters are required for special-purpose testing, state clearly their source and quality characteristics.
b. Phosphate buffer solution, 1.5N: Dissolve 207 g sodium dihydrogen phosphate, NaH
2PO4 /H18528H2O, in water. Neutralize to
pH 7.2 with 6 N KOH (¶ g below) and dilute to 1 L.
c. Ammonium chloride solution, 0.71N: Dissolve 38.2 g am- monium chloride, NH 4Cl, in water. Neutralize to pH 7.0 with KOH. Dilute to 1.0 L; 1 mL /H1100510 mg N.
d. Calcium chloride solution, 0.25N: Dissolve 27.7 g CaCl 2 in water and dilute to 1 L; 1 mL /H1100510 mg Ca.
e. Magnesium sulfate solution, 0.41N: Dissolve 101 g
MgSO4 /H185287H2O in water and dilute to 1 L; 1 mL /H1100510 mg Mg. f. Ferric chloride solution, 0.018N: Dissolve 4.84 g
FeCl3 /H185286H2O in water and dilute to 1 L; 1 mL /H110051.0 mg Fe. g. Potassium hydroxide solution, 6N: Dissolve 336 g KOH in about 700 mL water and dilute to 1 L. CAUTION: Add KOH to water slowly and use constant mixing to prevent excessive heat buildup. Alternately, use commercial solutions containing 30 to 50% KOH by weight.
h. Acid solutions, 1N: Add 28 mL conc H
2SO4 or 83 mL conc
HCl to about 700 mL water. Dilute to 1 L.
i. Alkali solution, 1N: Add 40 g NaOH to 700 mL water.

Dilute to 1 L.
j. Nitriﬁcation inhibitor: Reagent-grade 2-chloro-6-(trichlo- romethyl) pyridine (TCMP) or equivalent. 3*
k. Glucose-glutamic acid solution: Dry reagent-grade glucose and reagent-grade glutamic acid at 103°C for 1 h. Add 15.0 g glucose and 15.0 g glutamic acid to distilled water and dilute to 1 L. Neutralize to pH 7.0 using 6 N potassium hydroxide (¶ g above). This solution may be stored for up to 1 week at 4°C. l. Electrolyte solution (for electrolytic respirometers): Use manufacturer’s recommended solution.
m. Sodium sulﬁte solution, 0.025N: Dissolve 1.575 g Na
2SO3
in about 800 mL water. Dilute to 1 L. This solution is not stable; prepare daily or as needed.
n. Trace element solution: Dissolve 40 mg MnSO 4 /H185284H2O, 57 mg H 3BO3, 43 mg ZnSO 4 /H185287H2O, 35 mg (NH 4)6 Mo7O24, and 100 mg Fe-chelate (FeCl 3-EDTA) in about 800 mL water.

Dilute to 1 L. Sterilize at 120°C and 200 kPa (2 atm) pressure for 20 min.
* Formula 2533, Hach Chemical Co., Loveland, CO, or equivalent. N OTE: Some commercial formulations are not pure TCMP. Check with supplier to verify compound purity and adjust dosages accordingly.

BIOCHEMICAL OXYGEN DEMAND (5210)/Respirometric Method
10 BIOCHEMICAL OXYGEN DEMAND (5210)/Respirometric Method o. Yeast extract solution: 3 Add 15 mg laboratory- or pharma - ceutical-grade brewer’s yeast extract to 100 mL water. Make this solution fresh immediately before each test in which it is used. p. Nutrient solution:
3 Add 2.5 mL phosphate buffer solution
(3b), 0.65 mL ammonium chloride solution (3 c), 1.0 mL calcium chloride solution (3d), 0.22 mL magnesium sulfate solution (3 e),

0.1 mL ferric chloride solution (3 f), 1 mL trace element solution (3n), and 1 mL yeast extract solution (3 o) to about 900 mL water. Dilute to 1 L. This nutrient solution and those of ¶s n and o above are speciﬁcally formulated for use with the OECD method.3 (NOTE: A 10:1 concentrated nutrient solution can be made and diluted accordingly.)

4. Procedure
a. Instrument operation: Follow respirometer manufacturer’s instructions for assembly, testing, calibration, and operation of the instrument. N
OTE: The manufacturer’s stated maximum and
minimum limits of measurement are not always the same as the instrument output limits. Make sure that test conditions are within the limits of measurement.
b. Sample volume: Sample volume or concentration of organic chemicals to be added to test vessels is a function of expected oxygen uptake characteristics and oxygen transfer capability of the instrument. Small volumes or low concentrations may be required for high-strength wastes. Large volumes may be re- quired for low-strength wastes to improve accuracy.
c. Data recording interval: Set instrument to give data read- ings at suitable intervals. Intervals of 15 min t o 6 h typically are used.
d. Sample preparation:
1) Homogenization—If sample contains large settleable or ﬂoatable solids, homogenize it with a blender and transfer rep- resentative test portions while all solids are in suspension. If there is a concern for changing sample characteristics, skip this step.
2) pH adjustment—Neutralize samples to pH 7.0 with H
2SO4
or NaOH of such strength (5210D.3 h and i) that reagent quantity does not dilute the sample more than 0.5%.
3) Dechlorination—Avoid analyzing samples containing re- sidual chlorine by collecting the samples ahead of chlorination processes. If residual chlorine is present, aerate as described in ¶ 5) below or let stand in light for 1 to 2 h. If a chlorine residual persists, add Na
2SO3 solution. Determine required volume of
Na2SO3 solution by adding 10 mL 1 /H110011 acetic acid or 1 /H1100150 H2SO4 and 10 mL potassium iodide solution (10 g/100 mL) to a portion of the sample. Titrate with 0.025 N Na2SO3 solution to the starch-iodine end point (see Section 4500-Cl.B). Add to the neutralized sample a proportional volume of Na 2SO3 solution determined above, mix, and after 10 to 20 min check for residual chlorine. Re-seed the sample (see ¶ h below).
4) Samples containing toxic substances—Certain industrial wastes contain toxic metals or organic compounds. These often require special study and treatment.
3 5) Initial oxygen concentration—If samples contain dissolved oxygen concentrations above or below the desired concentration, agitate or aerate with clean and ﬁltered compressed air for about 1 h immediately before testing. Minimum and maximum actual DO concentrations will vary with test objectives. In some cases, pure oxygen may be added to respirometer vessels to increase oxygen levels above ambient.
6) Temperature adjustment—Bring samples and dilution water to desired test temperature ( /H110061°C) before making dilutions or transferring to test vessels.
e. Sample dilution: Use distilled water or water from other appropriate sources free of organic matter. In some cases, re- ceiving stream water may be used for dilution. Add desired sample volume to test vessels using a wide-tip volumetric pipet or other suitable volumetric glassware. Add dilution water to bring sample to about 80% of desired ﬁnal volume. Add appro- priate amounts of nutrients, minerals, buffer, nitriﬁcation inhib- itor if desired, and seed culture as described in ¶s f–h below.

Dilute sample to desired ﬁnal volume. The number of test vessels to prepare for each dilution depends on test objectives and number of replicates desired.
f. Nutrients, minerals, and buffer: Add sufﬁcient ammonia nitrogen to provide a COD:N:P ratio of 100:5:1 or a TOC:N:P ratio of 30:5:1. Add 2 mL each of calcium, magnesium, ferric chloride, and trace mineral solutions to each liter of diluted sample unless sufﬁcient amounts of these minerals are present in the original sample. Phosphorus requirements will be met by the phosphate buffer if it is used (1 mL/50 mg/L COD or ultimate BOD of diluted sample usually is sufﬁcient to maintain pH between 6.8 and 7.2). Be cautious in adding phosphate buffer to samples containing metal salts because metal phosphates may precipitate and show less toxic or beneﬁcial effect than when phosphate is not present. For OECD-compatible tests, substitute the nutrient, mineral, and buffer amounts listed in 5210D.3 p for the above nutrient/mineral/buffer quantities.
g. Nitriﬁcation inhibition: If nitriﬁcation inhibition is desired, add 10 mg 2-chloro-6-(trichloromethyl) pyridine (TCMP)/L sample in the test vessel. Samples that may nitrify readily in- clude biologically treated efﬂuents, samples seeded with biolog- ically treated efﬂuents, and river waters.
4 h. Seeding: See 5210B.4d for seed preparation. Use sufﬁcient amounts of seed culture to prevent major lags in the oxygen uptake reaction but not so much that the oxygen uptake of the seed exceeds about 10% of the oxygen uptake of the seeded sample.

Determine the oxygen uptake of the seeding material as for any other sample. This is the seed control. Typically, the seed volume in the seed control should be 10 times the volume used in seeded samples.
i. Incubation: Incubate samples at 20°C or other suitable temperature /H110061.0°C. Take care that the stirring device does not increase the temperature of the sample.

5. Calculations
To convert instrument readings to oxygen uptake, refer to manufacturer’s procedures.

Correct oxygen uptake for seed and dilution by the following equation:

C /H11005[A /H11002B(SA/SB)](1000/NA)
where:

C /H11005corrected oxygen uptake of sample, mg/L,
A /H11005measured oxygen uptake in seeded sample, mg,
BIOCHEMICAL OXYGEN DEMAND (5210)/Respirometric Method
11 BIOCHEMICAL OXYGEN DEMAND (5210)/Respirometric Method B /H11005measured oxygen uptake in seed control, mg,
SA /H11005volume of seed in Sample A, mL,
SB /H11005volume of seed in Sample B, mL, and
NA /H11005volume of undiluted sample in Sample A, mL.

6. Quality Control
The quality control practices considered to be an integral part of each method are summarized in Table 5020:I.

Periodically use the following procedure to check distilled water quality, instrument quality, instrument function, and ana- lytical technique by making oxygen uptake measurements using a mixture of glucose and glutamic acid as a standard check solution.

Adjust water for sample formulation to test temperature and saturate with DO by aerating with clean, organic-free ﬁltered air.

Protect water quality by using clean glassware, tubing, and bottles.

Prepare a test solution by adding 10 mL glucose-glutamic acid solution (3k); 6 mL phosphate buffer (3 b); 2 mL each of ammo- nium chloride (3 c), magnesium sulfate (3 e), calcium chloride (3d), ferric chloride (3 f), and trace element solution (3 n)t o approximately 800 mL water. Add 10 mg nitriﬁcation inhibitor (TCMP)/L. Add sufﬁcient seed from a suitable source as de- scribed in 5210D.4 h to give a lag time less tha n 6 h (usually 25 mL supernatant from settled primary efﬂuent/L test solution is sufﬁcient). Dilute to 1 L. Adjust temperature to 20 /H110061°C.

Prepare a seed blank by diluting 500 mL or more of the seed solution to 800 mL with distilled water. Add the same amount of buffer, nutrients, and TCMP as in the test solution, and dilute to 1 L. Adjust temperature to 20 /H110061°C.

Place test solution and seed blank solution in separate reaction vessels of respirometer and incubate fo r5da t 20°C. Run at least three replicates of each. The seed-corrected oxygen uptake after 5 d incubation should be 260 /H1100630 mg/L. If the value of the check is outside this range, repeat the test using a fresh seed culture and seek the cause of the problem.

7. Precision and Bias
a. Precision: No standard is available to check the accuracy of respirometric oxygen uptake measurements. To obtain labora- tory precision data, use a glucose-glutamic acid mixture (5210D.6) having a known theoretical maximum oxygen uptake value. Tests with this and similar organic compound mixtures have shown that the standard deviation, expressed as the coef- ﬁcient of variation, C
v, is approximately 5% for samples having
total oxygen uptakes of 50 to 100 mg/L and 3% for more
concentrated samples. 1,2 Individual instruments have different readability limits that can affect precision. The minimum re- sponse or sensitivity of most commercial respirometers ranges from 0.05 to 1 mg oxygen. Check manufacturer’s speciﬁcations for sensitivity of the instrument at hand.
b. Control limits: To establish laboratory control limits, per- form a minimum of 25 glucose-glutamic acid checks over a period of several weeks or months and calculate mean and standard deviation. If measured oxygen uptake i n5da t 20°C is outside the 260 /H1100630 mg/L range, re-evaluate procedure to identify source of error. For other samples, use the mean /H110063 standard deviations as the control limit.
c. Working range and detection limits: The working range and detection limits are established by the limits of each commercial instrument. Refer to manufacturer’s speciﬁcations.

8. References

1. Y OUNG, J.C. & E.R. B AUMANN. 1976. The electrolytic respirometer— Factors affecting oxygen uptake measurements. Water Res. 10:1031.

2. Y OUNG, J.C. & E.R. B AUMANN. 1976. The electrolytic respirometer— Use in water pollution control plant laboratories. Water Res. 10:1141.

3. O RGANIZATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT. 1981.

Method 5.2, Annex V, Part C in OECD Guidelines for Testing of Chemicals. OECD, Paris, France.

4. Y OUNG, J.C. 1973. Chemical methods for nitriﬁcation control. J.

Water Poll. Control Fed. 45:637.

9. Bibliography
HEUKELEKIAN, H. 1947. Use of direct method of oxygen utilization in waste treatment studies. Sew. Works J. 19:375.

CALDWELL, D.H. & W.F. L ANGELIER. 1948. Manometric measurement of the biochemical oxygen demand of sewage. Sew. Works J. 20:202.

GELLMAN,I .&H .H EUKELEKIAN. 1951. Studies of biochemical oxidation by direct methods. Sew. Ind. Wastes. 23:1267.

JENKINS, D. 1960. The use of manometric methods in the study of sewage and trade wastes. In Waste Treatment, p. 99. Pergamon Press, Oxford, U.K.

MONTGOMERY, H.A.C. 1967. The determination of biochemical oxygen demand by respirometric methods. Water Res. 1:631.

CEDENA, F., A. D ROHOBYCZAR, M.I. B EACH &D .B ARNES. 1988. A novel approach to simpliﬁed respirometric oxygen demand determina- tions. Proc. 43rd Ind. Waste Conf. , Purdue Univ., Lafayette, Ind.

BIOCHEMICAL OXYGEN DEMAND (5210)/Respirometric Method
12 BIOCHEMICAL OXYGEN DEMAND (5210)/Respirometric Method SOP AMBL-105-D Page 1 of 7
Standard Operating Procedure
AMBL-105-D

Total Suspended Solids by Gravimetric
Determination

METHOD SUMMARY
This SOP describes the procedure for measuring total suspended solids in water and wastewater. This method is based on Method 2540 D of Standard Methods for the Examination of Water and Wastewater, 24th Edition, 2023.

ENVIRONMENTAL HEALTH AND SAFETY
Hazards Assessment: This method involves the use of a convection oven and optionally a muffle furnace, the handling of natural waters or untreated wastewaters that potentially contain pathogenic organisms. The specific hazards associated with this method are as follows.

Burns: Burns to the hands or arm are possible if the sides of the convention oven or muffle furnace are touched when placing the sample into or removing it from the oven or furnace. Burns will also occur if the hot porcelain evaporating dish itself is touched.

Biological Hazard: The presence of pathogenic organisms must be assumed, regardless of the water sample source. Natural waters, sewage and wastewater all contain bacteria, funguses, parasites, and viruses that can lead to intestinal or other infections, including but not limited to diarrhea, fever, nausea, cramps, vomiting, headaches, conjunctivitis (pink eye) and Hepatitis A.

Safety Equipment and Engineering Controls: This method requires that you wash your hands with soap when finished handling samples and that an eye wash station be located nearby.

Personal Protective Equipment (PPE): This method requires the use of the following PPE.

Gloves (nitrile, PVC or neoprene)
Prepared: 12/27/2017
Prepared by: Terry E Baxter
Last Reviewed: 7/1/2023
Last Revised: 7/9/2023
Revised by: Terry E Baxter

SOP AMBL-105-D Page 2 of 7
Safety goggles or glasses
Laboratory coat
Analysis-derived Wastes and Disposal:

Waste Generated Hazardous
( Y / N) Disposal
This procedure generates a
dried solid residue on the
surface of a glass-fiber filter.

N
A solid wastewater solids residue is
considered desiccated and to have
heat-killed (> 71C) bacteria. The filter
and solid residue may be disposed in
the laboratory trash.

METHOD DESCRIPTION

1.0 Introduction and Applicability
Total suspended solids or TSS is the measure of the undissolved solid matter in a water that remains on the surface of a filter after all the water has been evaporated. Suspended solids affect water quality by making it unfit or unsafe to drink, aesthetically unacceptable for recreational use and aquatic habitats, and unsuitable for use in many industrial or other applications. A known volume of a well-mixed sample is filtered through a standard glass-fiber filter, collecting the solid residue on the surface of the filter. The filter and residue are evaporated to a constant weight condition in an oven maintained at a temperature of 103-105C. The mass of the dried residue is determined and used to calculate the concentration of total suspended solids in the sample.

This method is applicable for measurement of total suspended solids in all natural waters, in raw, process and treated agricultural, municipal and industrial wastewaters. This method is not considered applicable to wastewater slurries behaving as a Newtonian fluid, non-Newtonian fluids or treated drinking water.

2.0 Apparatus
a. Glass-fiber filter, with a 47 mm diameter, nominal pore size  2.0 µm and  1.0 µm, and no binders.
b. Graduated cylinder, Class A
c. Wide-bore pipet, Class B
d. Forceps capable of lifting and holding a filter without tearing or puncturing it.
e. Filter pans, aluminum or other inert material, to hold filters. f. Convection oven operated at 103-105C for drying samples to a constant weight condition.

SOP AMBL-105-D Page 3 of 7
g. Muffle furnace operated at 550  50C (if volatile solids will be determined).
h. Desiccator containing a desiccant that responds (color change) to moisture or a hygrometer that measures moisture.
i. Analytical balance capable of weighing to the nearest 0.1 mg or less. j. Magnetic stirrer and stir bar (optional).
k. Blender or homogenizer (optional)
l. Beaker, low-form Class B or Class A having a volume sufficient enough to fully contain the sample and prevent sample loss from spillage or splattering when mixing.
m. Filtration funnel assembly for a 47 mm size diameter filter. n. Vacuum suction flask, 1000 mL capacity.
o. Vacuum hose (thick-walled)
p. Vacuum trap

3.0 Reagents
a. Reagent water, deionized/reverse osmosis (DI/RO) or distilled water (DW)

4.0 Procedure
a. Read Method 2540D Total Suspended Solids Dried at 103-105C (Standard Methods).
b. Prepare a glass-fiber filter by placing and centering a filter disk onto the filter support screen of the filtration apparatus and attach the funnel.

Apply a low to moderate vacuum and rinse the filter with three successive volumes of 30 mL reagent water. Leave the vacuum on until all traces of water have been removed from the filter. Wet filters can adhere to the filter pan during the drying process and cause filter fibers to be torn from the filter when lifted for weighing. Remove the hose and turn off the vacuum. Use forceps to carefully remove the filter from the filtration apparatus support screen by holding and slowly lifting the filter only by the outer edge of the filter. Transfer the filter to a filter pan. Place the filter and pan into a drying oven operated at a temperature of 103-105C. Dry the filter at this temperature for no less than 60 minutes if measuring only total suspended solids or, if volatile suspended solids will be determined (see SOP 105E) place the filter in a muffle furnace at a temperature of 550  50C for no less than 15 minutes. In a desiccator, cool the dried filter to room temperature.

Remove the filter from the filter pan, weigh and record its weight - this is the tared weight of the filter. Replace the filter in the filter pan and store it in a desiccator until used.
c. Equilibrate the sample’s temperature to that of the room’s temperature and use a pipet or graduated cylinder to transfer a volume of well-mixed SOP AMBL-105-D Page 4 of 7
sample onto the filter with the vacuum applied. Use a graduated cylinder for samples having solids that clog the wide bore pipet tip. Select a sample volume that will result in a dried residue ranging from 2.5 to 200 mg. Avoid filtration times exceeding 10 minutes. Rinse the entire surface area of the exposed filter with three successive volumes of 10 mL reagent water. Allow the water to completely drain between each rinsing and leave the vacuum on until all traces of water have been removed from the filter. Remove the hose and turn off the vacuum.

Carefully remove the filter from the filtration apparatus support screen using forceps by lifting and holding the filter only by the clean outer edge without solid residue. Transfer the filter to a filter pan. d. Dry the filter with solid residue in a convection oven at a temperature of 103-105C for no less than 60 minutes. Drying samples overnight is acceptable and an appropriate procedural step for the AMBL. In most circumstances, this ensures that constant weight has been achieved. e. Remove the filter pan containing the filter from the oven, place it in a desiccator and cool it to room temperature. Carefully remove the filter from the filter pan using the forceps without touching the dried solid residue and weigh it. Record this as the first 103C weight. f. Repeat the drying cycle for no less than 60 minutes, and again cool, weigh and record the second 103C weight.
g. Calculate the weight change between the first and second weights, and if the change is >0.5 mg, repeat the drying cycle until the change in weight between the final weight and the previous weight is 0.5 mg.

Record and use this final 103C weight.

4.0 Calculation and Reporting
a. Calculate the concentration of total suspended solids Total Suspended Solids, as mg TSS/L (𝐴−𝐹) × 1,000
𝑆
where A = final 103C weight of the dried residue + the tared filter, mg,
F = tared filter weight, mg, and
S = mL of sample volume.
b. Identify any sample that yields a residue mass < 2.5 mg or > 200 mg and report the results as an “estimate” because the mass has exceeded the criteria of this analysis.

SOP AMBL-105-D Page 5 of 7
c. Report as follows:

Calculated Range
(mg/L)
Reported to
nearest (mg/L)
< 50 mg/L 1 mg/L
50 – 99 mg/L 5 mg/L
100 – 4999 mg/L 10 mg/L
> 5000 mg/L 50 mg/L

d. Once the calculated TSS value exceeds 10,000 mg/L, it is recommended to measure solids using SOP 105 F.

5.0 Quality Assurance and Quality Control
a. Sample and Filter Handling
The suspended solid material in a liquid medium sample is considered relatively non-homogeneous. The sub-sampling of a non-homogenous material prior to the filtration step as well as how the filter is handled throughout the procedure can introduce variability and are thus considered part of this method’s quality assurance and quality control practices. 1) A well-mixed sample is essential for minimizing the non- homogeneous nature of the suspended material. Mixing the sample in a beaker using a magnetic stirrer is generally preferred as long as the mixing regime provided consists of a vertical mixing component as well as the horizontal rotation of the sample. A sample can also be well-mixed by hand, either by inverting a closed sample container multiple times or stirring the sampling with a stirring rod or the pipet in a way that can fully agitate the sample. When using a pipet, it is best to use a pipet bulb that does not leak air so that sample does not enter the pipet while agitating the sample. However, mixing by hand requires that the sample be agitated prior to each time a sample aliquot is taken.
2) Improper alignment and placement of the filter on the support grid can cause a tear or crease in the filter when the filtration funnel is placed over the filter, or in the worst case, leave a portion of the porous support grid exposed. A tear or crease weakens the edge of the filter and can result in the loss of filter material when lifting or moving the filter. Realigning a filter that has a tear, will allow some sample and solids to pass through the tear and thus bypass the filter.

An exposed portion of the support grid would also allow some sample and solids to bypass the filter. Proper alignment of the filter on the support grid should be visually verified before placing the filtration funnel over the filter.

SOP AMBL-105-D Page 6 of 7
3) A filter is picked up using forceps that will not puncture or tear the filter, and should always be held by placing the forceps near the edge of the filter that remains free of solid material residue. Forceps that touch the solids residue on the filter can pick up some of the residue and transfer it to the next filter that is handled. Make sure that the forceps are clean prior to handling any filter.
4) Extended filtration times can occur when the sample solids concentration is high and too much sample volume has been used.

A solids particle-size distribution that is predominantly and only just somewhat larger than the filter’s effective pore size can cause also extend filtration time because the filter’s pore will become clogged more rapidly. Regardless, extended filtration times can lead to the filter adhering to the support grid and causing the filter to tear or lose fibers from the bottom of the filter when the filter is lifted. One effect that occurs during extended filtration times is the formation of ice within the support grid along the bottom of the filter since the air temperature beneath the filter will decrease because of the vacuum.

Always lift the filter from the support screen after the vacuum has been turn off and the vacuum line removed and lift slow enough so a filter adhered to the support grid can be detected and measures to prevent tearing fibers from the bottom of the filter may be taken. If freezing is expected, wait a few minutes to allow the ice to thaw before removing the filter.
5) A damp filter, with or without solid residue, that is placed into the weighing pan tends to adhere to the pan’s flat surface that results in a visible loss of filter fibers on the pan when the filter is lifted.

Ensuring that the rinse water is removed from the solids and filter to the extent possible before the vacuum is turned off and the vacuum hose removed can prevent this from happening. Following this practice is also beneficial for potentially reducing the number of drying cycles.
6) Verifying that the filter and solid material residue have achieved a constant weight is critical. Constant weight is defined as having been achieved if the mass change between two subsequent drying cycles is less than 0.5 mg. Although many samples can achieve a constant weight condition after one drying cycle, it requires no less than one additional drying cycle to verify that this has been achieved. The exception to this is when a drying time study is conducted for the specific sample and suspended solids type that demonstrates overnight drying alone can achieve constant weight. An AMBL study conducted in 2016 demonstrated that overnight drying achieved a constant weight for mixed liquor suspended solids.

SOP AMBL-105-D Page 7 of 7
b. Data Quality Assessment and Corrective Actions
Assessing data quality and method performance is done by preparing and analyzing various quality control samples with some defined frequency. The results of these QC samples are then evaluated against preferably lab-specific performance criteria or against criteria considered acceptable performance indicators across multiple labs. 1) Analyze a method blank (a clean, dried, and tared filter) throughout the entire process with each batch of 20 or fewer samples. If a single sample is being analyzed, a method blank must also be analyzed.

Evaluate the method blank result against the AMBL-specific criteria of <0.2 mg mass difference between two subsequent drying cycles. 2). Analyze at least one sample in duplicate with each batch of 20 or fewer samples. If a single sample is being analyzed, this sample must be analyzed in duplicate. Evaluate sample duplicates by calculating relative percent difference (RPD) as follows.

𝑅𝑃𝐷, % =|𝑆𝑎𝑚𝑝𝑙𝑒−𝐷𝑢𝑝𝑙𝑖𝑐𝑎𝑡𝑒 𝑆𝑎𝑚𝑝𝑙𝑒|
(𝑆𝑎𝑚𝑝𝑙𝑒+𝐷𝑢𝑝𝑙𝑖𝑐𝑎𝑡𝑒 𝑆𝑎𝑚𝑝𝑙𝑒)/2× 100

Evaluate the RPD value against the AMBL-specific criteria of < 5%. 3) Analyze one laboratory-fortified blank and laboratory-fortified blank duplicate sample set (LFB/LFBD) for each 20 samples analyzed, not including method blanks or duplicate samples. Prepare a LFB sample for total suspended solids by weighing 100 mg Celite 545 (record the actual weight) to the nearest 0.1 mg. Suspend in distilled water to a volume of 1 liter. Measure the total suspended solids of this standard in duplicate. The RPD of the LFB/LFBD analyses should not exceed an absolute value of 10%. An AMBL-specific criteria has not yet been established.

6.0 References

1. American Public Health Association, American Water Works Association, Water Environment Federation. Lipps WC, Braun- Howland EB, Baxter TE, eds. Standard Methods for the Examination of Water and Wastewater. 24th ed. Washington DC: APHA Press; 2023.

Treatment Plant Connection
Capacity Charges
Flow BOD TSS
Current 563000 gal/d 1750 lb/day 1460 lb/day
2027 886000 gal/d 2200 lb/day 2300 lb/day
Amount of Increase 323000 gal/d 450 lb/day 840 lb/day
Rate 2.90$ 358.70$ 225.50$ Fee 936,700.00$ 161,415.00$ 189,420.00$ Trunk Line Capacity Charge
Current 850000 gal/d
2027 1329000 gal/d
Amount of Increase 479000 gal/d
Rate 1.26$
Fee 603,540.00$
Total Due 1,891,075.00$
Exhibit "C"
Application of Treatment Plant Capacity Connection Charges and Trunk Line Capacity Charge to 2027 Capacity Increase, If Paid Prior to July 30, 2026

Visalia City CouncilJune 15, 2026

Visalia City Council
June 15, 2026