pH stabilization during testing of lethality of wastewater effluent to rainbow trout: front matter
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Lisa Taylor
Manager, Method Development & Applications Section
Environmental Science and Technology Centre
Environment Canada
335 River Road
Ottawa, Ontario
K1A 0H3
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Abstract
This document provides detailed techniques, conditions, and guidance for the pH stabilization of wastewater effluent samples. The procedure described herein must be used in conjunction with the explicit instructions given in the reference method EPS 1/RM/13 “Biological Test Method: Reference Method for Determining Acute Lethality of Effluents to Rainbow Trout” (Environment Canada, 2000). This procedure is not stand-alone; it is an add-on to the rainbow trout method for acute lethality.
In many wastewater effluent samples, the carbon dioxide (CO2) content may be elevated as a result of high biological activity. Aeration of these samples may cause the pH to rise because of a loss of CO2, and this change in pH can alter the toxicity of the ammonia present in the wastewater effluent sample. The purpose of pH stabilization is to replace the CO2 lost due to aeration in order to maintain the pH throughout the test at the same levels found in the initial samples.
In order to use this add-on procedure, the wastewater effluent sample must meet three conditions: (i) total ammonia must be measured on all wastewater effluent samples submitted for testing with EPS 1/RM/13, (ii) the wastewater effluent must have failed an acute lethality test using rainbow trout (EPS 1/RM/13) on a previously collected sample, and (iii) pH stabilization techniques may only be used when the un-ionized ammonia concentration present in the 100% wastewater effluent sample does not equal or exceed 1.25 mg/L at 15°C or when the total ammonia concentration does not equal or exceed the maximum total ammonia concentration (y) in mg/L determined using the following formula and the initial pH of the wastewater effluent sample at 15°C:
y = 1.25 x (10(9.564136638-pH) + 1)
This procedure document describes three pH stabilization techniques that can be used as an add-on to EPS 1/RM/13: (i) CO2 injection; (ii) Recycling; and (iii) pH Controller.
This pH stabilization procedure applies to both single-concentration tests and multi-concentration tests to determine the median lethal concentration (LC50). Instructions are included on the apparatus setup, observations and measurements to be made, and maintaining pH control throughout the test. Validity criteria for this add-on procedure are outlined, and these must be met in addition to those outlined in EPS 1/RM/13.
Foreword
The three techniques for stabilizing the pH of a wastewater effluent during an acute lethality test function as add-on procedures to the reference method, EPS 1/RM/13; the technique chosen must be used in conjunction with this reference method for measuring and assessing the toxic effect(s) of wastewater effluent on rainbow trout. It may only be used when the test sample has met the three conditions outlined within this document; these conditions pertain to the measurement of total ammonia on all wastewater effluent samples submitted for toxicity testing, the failure of the reference method on a previously collected sample of wastewater effluent, and the amount of un-ionized ammonia in the sample of wastewater effluent to be tested.
This add-on procedure outlines explicit sets of instructions and conditions to be used with EPS 1/RM/13 and are applied only to wastewater effluent samples as defined in this document.
Terminology
The words defined in this section are italicized when first used in the body of the report according to the definition. All definitions are given in the context of the procedures in this report, and might not be appropriate in another context.
Grammatical Terms
Can is used to mean “is (are) able to.”
May is used to mean “is (are) allowed to.”
Must is used to express an absolute requirement.
Should is used to state that the specified condition or procedure is recommended and ought to be met if possible.
Technical Terms
Acute means happening within a short period of time (≤96-h for the rainbow trout acute lethality test).
Alkalinity means the acid-neutralizing capacity of water, reported as mg/L as calcium carbonate (CaCO3) (see also American Public Health Association (APHA) et al., 2005).
Ammonia means total ammonia [NH3 + NH4, as nitrogen (N)], un-ionized ammonia (NH3, as N) and ionized ammonia (NH4+, as N). The percentage of un-ionized ammonia (NH3) in total ammonia is determined by pH and temperature. The following formulae are used to calculate the fraction of un-ionized (NH3) and ionized (NH4+) ammonia. Since NH3 = 1/(1 + 10 pK - pH) and NH4+ = 1/(1 + 10 pH - pK), and total ammonia = NH3 + NH4+, the concentration of un-ionized ammonia (assuming a pK of 9.56 at 15 ºC) is calculated as: un-ionized ammonia = (total ammonia) × [1/(1 + 10 pH - pK)] (United States Environmental Protection Agency (USEPA), 1999).
BOD means biological oxygen demand and refers to the amount of oxygen consumed when organic matter in a volume of water is biodegraded (see also APHA et al., 2005).
Buffering capacity is the ability of water to maintain a stable pH which is controlled by the amount of carbonate ions (alkalinity) present in water.
Control means, in this test method, a treatment that duplicates all the conditions and factors that might affect the results, except the specific condition that is being studied. In an aquatic toxicity test, the control must duplicate all conditions of the exposure treatment(s), but must contain no test material. The control is used to determine the absence of toxicity due to basic test conditions (e.g., temperature, quality of dilution water, health of test organisms, or effects due to their handling).
Control/dilution water means water that is used for diluting the sample of effluent, and for the control test.
Dead fish are fish in which all visible signs of movement or other activity have ceased (see also Section 4.5 of EPS 1/RM/13).
Effluent is any liquid waste (e.g., industrial, municipal) discharged to the aquatic environment. See definition of wastewater effluent for the meaning of a specific category of effluent.
Hardness means the concentration of cations in water that will react with a sodium soap to precipitate an insoluble residue. In this method, hardness means a measure of the concentration of calcium (Ca++) and magnesium (Mg++) ions in water, expressed as mg/L calcium carbonate (CaCO3) (see also APHA et al., 2005).
LC50 (median lethal concentration) means the concentration of effluent in water that is estimated to be lethal to 50% of the test organisms with a 96-hour exposure period. The LC50 and its 95% confidence limits are derived by statistical analysis of percent mortalities in several test concentrations, after a fixed period of exposure.
Lethal means causing death by direct action.
pH is the negative logarithm of the activity of hydrogen ions in gram equivalents per litre. The pH value expresses the degree or intensity of both acidic and alkaline reactions on a scale from 0 to 14, with 7 representing neutrality, numbers less then 7 signifying increasingly greater acidic reactions, and numbers greater than 7 indicating increasingly basic or alkaline reactions.
pH i (initial pH) refers to the pH as measured on composite 100% sample at 15 ± 1°C before any aeration of the test solution at the lab.
pH stabilized test means the EPS 1/RM/13 test method with a pH stabilization technique applied on a wastewater effluent sample.
Reference method means a specific biological test method for performing a toxicity test, i.e., a toxicity test method with an explicit set of instructions and conditions which are described precisely in a written document. Unlike other multi-purpose (generic) biological test methods published by Environment Canada, the use of a reference method is frequently restricted to testing requirements associated with specific regulations; testing to assess whether there has been a violation of the General Provisions of the Canadian Fisheries Act.
Static means toxicity tests in which the test solutions are not renewed during the test.
Sublethal means deleterious to fish, but below the level that directly causes death of fish within the 96-hour test period.
Toxicity means the inherent potential or capacity of a substance to cause deleterious effect(s) on fish. The effect(s) may be lethal or sublethal.
Wastewater means a mixture of liquid wastes primarily composed of domestic sewage that can also include other liquid wastes from industrial, commercial and institutional sources.
Wastewater effluent means untreated or treated wastewater that is released from the outfall(s) of a wastewater system, excluding combined sewer overflows from the wastewater system.
Wastewater system means any works for the collection or treatment and release of wastewater or any part of such works.
Acknowledgements
This document was prepared by Lesley Novak and Keith Holtze (Stantec Consulting Ltd., Guelph, Ontario). Rick Scroggins (Chief, Biological Methods Division, Environment Canada, Ottawa, ON) was the Scientific Authority for the project, providing strategic direction, detailed review comments, and technical assistance throughout the work. Lisa Taylor (Manager, Method Development and Applications Section, Environment Canada, Ottawa, ON) provided technical guidance and review comments. We would like to thank Graham van Aggelen and Grant Schroeder (Environment Canada, Pacific and Yukon Laboratory for Environmental Testing, North Vancouver, BC), Julie Schroeder and Richard Chong-Kit (Ontario Ministry of the Environment, Toronto, ON), and Garth Elliott and Nancy Kruper (Environment Canada, Prairie and Northern Laboratory for Environmental Testing, Edmonton, AB) for providing the standard operating procedures for the pH stabilization procedures, and technical advice and review comments on previous versions. Thanks also to Ken Doe and Paula Jackman (Environment Canada, Altantic Laboratory for Environmental Testing, Moncton, NB), and Brian Walker and Manon Harwood (Environment Canada, Quebec Laboratory for Environmental Testing, Montreal, QC) for their technical input and comments. All of the above laboratories also participated in the validation of the three pH stabilization techniques for application to wastewater effluent.
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