pH stabilization during testing of lethality of wastewater effluent to rainbow trout: chapter 1


Section 1: Introduction

In 1990, Environment Canada published a biological test method for conducting acute lethality tests with rainbow trout: Reference Method (RM) for Determining Acute Lethality of Effluent to Rainbow Trout (EPS 1/RM/13) (EC, 2000). The method (revised in 2007) was developed specifically for determining the acute lethality of effluent, and has been used across Canada by the federal, provincial, and territorial governments in the monitoring and control of industrial and wastewater effluent.

The test is conducted at 15 ± 1°C for 96 hours under static conditions (i.e., no renewal of test solution). Tests can be conducted using the full-strength (100%) wastewater only, or as a multiple concentrations (e.g., 100%, 50% etc.) to determine the LC50 (the concentration of wastewater sample that is estimated to be lethal to 50% of the animals exposed to that concentration within a defined period of exposure). Under most existing provincial and federal regulations, a sample is considered to “fail” the acute lethality test if >50% rainbow trout mortality is observed in the full-strength wastewater.

Aeration of both the control and test solutions at a rate of 6.5 ± 1 mL/min · L is a requirement of this test method. This aeration rate is sufficient to maintain the dissolved oxygen concentration in the control solution within 70% to 100% of the oxygen saturation value. The aeration rate is kept to a minimum, however, because excessive aeration of the wastewater can increase the rate of pH change and the removal of volatile compounds (ESG, 2002).

Aeration of wastewaters during acute lethality testing may cause the pH to rise from the equilibration of carbon dioxide (CO2) partial pressure in the wastewater with that in the atmosphere. The loss of CO2 due to aeration causes a shift in the carbonate buffering system of an effluent, and this leads to the rise in pH.

In many wastewater effluent samples, the CO2 content may be artificially elevated as a result of high biological activity, or from wastewater acidification prior to discharge (Mount and Mount, 1992). Any change in wastewater pH during an acute lethality test may affect mortality if the toxicity of the substance responsible is pH-dependent.

Ammonia, which could be of concern in wastewater effluent, would be one such example of a pH-dependent toxicant. Ammonia toxicity is attributable to the free or un-ionized (NH3-N) form as opposed to the ionized species. The relative concentration of un-ionized ammonia increases with increases in pH and water temperature. Depending on the initial pH of the full-strength wastewater and the magnitude of the upwards pH drift during testing, concentrations of un-ionized ammonia that were below lethal levels at test initiation, could increase sufficiently during testing to cause rainbow trout mortality by test completion.

To address the potential for residual ammonia toxicity in a wastewater effluent due to pH drift, Environment Canada has standardized three pH stabilization techniques for the control of pH during rainbow trout acute lethality testing. These pH stabilization techniques are add-on procedures to the EPS 1/RM/13 test method, however, a pH stabilized test can only be performed if three conditions have been met as outlined in Sections 1.1, 1.2 and 1.3.

Additional supporting background information on the use of these pH stabilization techniques is provided in “Supplementary Background and Guidance for Investigating Acute Lethality of Wastewater Effluent to Rainbow Trout” (EC, 2008).

1.1 Condition #1 - Total Ammonia Measurement

Total ammonia (in mg/L) must be measured on all wastewater effluent samples submitted for toxicity testing using EPS 1/RM/13. This measurement will be used to determine if pH stabilization is appropriate for subsequent samples. This concentration of total ammonia is used in the calculation of un-ionized ammonia at the initial pH (pH i)Footnote 1 of the effluent at 15°C (refer to Condition #3, Section 1.3).

1.2 Condition #2 - Failure of Acute Lethality Reference Method EPS 1/RM/13

The techniques described herein for the pH stabilization of wastewater effluent must only be used when it has been shown that a previously collected sample of wastewater effluent from the same source failed the rainbow trout acute lethality test (EPS 1/RM/13; i.e., > 50% mortality)Footnote 2.

1.3 Condition #3-Maximum Ammonia Concentration

The procedures described herein 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)

These maximum values for ammonia are set to pre-screen those wastewaters that would result in rainbow trout mortality regardless of the pH drift observed during the acute lethality test. In other words, pH stabilization techniques are not appropriate if the ammonia concentration is already sufficiently high to cause rainbow trout mortality at the start of the acute lethality test. If this maximum un-ionized ammonia value is exceeded, it clearly identifies that a wastewater effluent is not of a quality where the pH drift phenomenon would be a consideration (i.e., ammonia is already at an acutely lethal concentration prior to testing). For additional information and supporting rationale please refer to Environment Canada (2008).

Given that “total ammonia” = NH3 + NH4+, the un-ionized ammonia concentration in mg/L must be calculated using the following formula (United States Environmental Protection Agency (USEPA), 1999):

Un-ionized ammonia = (total ammonia) × [1/(1 + 10 pK - pH)]

where:

pK is 9.56 at 15 ºC; pH is the initial pH of the wastewater effluent at 15 ºC; and total ammonia is in mg/L as measured for Condition #1, Section 1.1.

1.4 Overview of pH Stabilization Techniques

Three techniques can be used for pH stabilization during rainbow trout acute lethality testing of wastewater effluent samples when the conditions for use of pH stabilization are met:

  1. CO2 Injection,
  2. Recycling, and
  3. pH Controller.

Regardless of the technique selected, application of a pH stabilization procedure with an acute lethality test using rainbow trout will require hands-on training prior to testing an actual wastewater effluent sample. Some experimentation will also likely be required with each individual sample, since the specific water chemistry will vary among (or even within) wastewater systems.

In addition to specific test requirements for pH stabilization techniques, all method requirements and procedures for EPS 1/RM/13 must be followed during the conduct of the tests. The rationale behind each technique is to replace the CO2 lost during test aeration in order to maintain the pH at initial pH of the sample (pH i). The techniques are not intended to add more CO2 than is already present in a wastewater effluent. A detailed description of each pH stabilization technique is provided in Section 2.

Page details

Date modified: