pH stabilization during testing of lethality of wastewater effluent to rainbow trout: chapter 2

Section 2: Procedure Options for Conducting pH Stabilization of Wastewater Effluents

• 2.1 General Requirements
  • 2.1.1 Observations and Measurements
  • 2.1.2 Chlorinated Wastewater Samples
  • 2.1.3 Test Validity Criteria
• 2.2 pH Stabilization Using the CO₂ Injection Technique
  • 2.2.1 Apparatus for Delivering CO₂ Mixture
  • 2.2.2 Estimating Percent CO₂ Mixture Required to Stabilize pH
  • 2.2.3 Setting Flow Rates for CO₂ Mixture and Laboratory Air
  • 2.2.4 Controlling pH Drift
• 2.3 pH Stabilization Using the Recycling Technique
  • 2.3.1 Recycling Technique Setup
  • 2.3.2 Controlling pH Drift
• 2.4 pH Stabilization Using the pH Controller Technique
  • 2.4.1 Regulator/Solenoid Assembly
  • 2.4.2 pH Controller
  • 2.4.3 Controlling pH Drift

This section provides details for conducting each pH stabilization technique with wastewater effluent:

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

In the pH stabilization test, the pH of the sample is controlled at the level measured at test initiation (pH i) using one of the three techniques set out below. The pH stabilization procedure does not supercede the existing acute lethality test method using rainbow trout (EC, 2000), but describes “add-on” techniques. All tests must meet the requirements and procedures outlined in EPS 1/RM/13. However, additional monitoring (Section 2.1) and reporting (Section 3) requirements are mandatory with these pH stabilization techniques.

2.1 General Requirements

The pH stabilization procedures apply when the tests from EPS 1/RM/13 [Section 5 - single concentration test; Section 6 - multi-concentration (LC50) test] are performed. In either case, the highest concentration tested is the 100% wastewater effluent.

Prior to testing for regulatory purposes, some preliminary investigations may be required with each wastewater sample, since the specific effluent chemistry will vary among (or even within) facilities. For example, wastewater samples or control/dilution water with low alkalinity or low hardness may be susceptible to significant shifts in pH due to minimal buffering capacity, and therefore, require less CO₂. The data generated during the development of the CO₂ Injection and pH Controller techniques indicated that both could be successfully applied to wastewater effluent samples without any significant declines in pH. Difficulties encountered in control/dilution waters with low buffering capacity can be avoided by ensuring each test solution and the control is injected with only enough CO₂ to maintain a stable pH. This ensures that a control solution with low buffering capacity will likely receive less CO₂ than a wastewater test solution with a higher buffering capacity. This approach reduces the chances of control mortality that could result if excess CO₂ were to be added, while still meeting the objective of pH stabilization, which is to maintain the initial pH of a sample.

For the CO₂ Injection or pH Controller techniques, tests should be conducted in either glass aquaria or in non-toxic containers (e.g., polyethylene, polypropylene containers). When using the Recycling technique, glass aquaria are recommended due to the need for specially adapted lids required to control the headspace above the test solution.

All solutions must be aerated with oil-free compressed laboratory air (lab air) throughout the test, at a controlled rate of 6.5 ± 1 mL/min · L. All solutions for tests must be prepared before aeration is started. Upon preparation of the test solutions, all solutions must be aerated for 30 minutes at 6.5 ± 1 mL/min · L. Stabilization of pH must start when aeration is initiated. After 30 minutes aeration, the concentration of dissolved oxygen must be measured in at least the highest test concentration (normally 100% effluent). If (and only if) oxygen in the highest test concentration is <70% or >100% of air saturation, then aeration (i.e., before exposure of fish) of all solutions including the control(s) must be continued at 6.5 ± 1 mL/min · L. This period of aeration must be restricted to the lesser of 90 additional minutes and attaining 70% saturation in the highest test concentration (or 100% saturation if super-saturation is evident). Immediately thereafter, fish must be placed randomly in each test solution and the test must be initiated, regardless of whether 70 to 100% saturation was achieved in all test solutions.

Environment Canada (2000) requires that compressed air be bubbled through a clean air stone. For the CO₂ Injection technique, air stones must be used in the delivery of the CO₂ mix. For the Recycling and pH Controller techniques, air stones must be used in the delivery of laboratory air. For the pH Controller technique, a glass pipette is highly recommended for use in the delivery of the CO₂ gas. The use of a glass pipette in the delivery of CO₂ gas in the pH Controller technique provides better control of the amount of CO₂ gas that is delivered to the sample when the controller is activated.

Test results may be confounded or difficult to interpret in cases when there is a difference in pH (> 0.2 pH units) between the 100% wastewater effluent sample and laboratory dilution water used to prepare exposure solutions for a multiple concentration test. The pH of each effluent concentration (i.e., 100, 50, 25, 12.5, 6.25%) must be maintained at the pH value measured at test initiation (before any aeration is started) in each individual exposure concentration and the control. However, there may be a gradient of pH values observed during testing that could result in a non-dose related response. In this case (i.e., when mortality is observed in diluted effluent concentrations, but not in the 100% concentration), an LC50 should not be calculated. Results for the 100% wastewater effluent sample will still be considered acceptable, provided that all other validity criteria are met (see Section 2.1.3).

2.1.1 Observations and Measurements

In addition to the observations and measurements described in EPS 1/RM/13 (e.g., temperature, dissolved oxygen, colour, turbidity, odour, and floating or settling solids), the laboratory must measure the pH, total ammonia, and hardness in each wastewater effluent sample. Total ammonia must be measured to at least two decimal places. Alkalinity must be measured if the CO₂ Injection technique is to be used. Measurements must be taken only after the contents of all containers have been thoroughly mixed and the temperature of the sample has been adjusted to 15 ± 1 °C. These parameters must be measured in the full strength sample after sub-samples (e.g., aliquots of a sample divided between two or more containers) have been combined. When a multiple concentration (LC50) test is conducted, pH, ammonia and hardness must be measured in each test solution; if the CO₂ Injection technique is used, alkalinity must be measured in the 100% wastewater effluent sample.

Before any aeration of the test solutions, the un-ionized ammonia concentration must be calculated using the measurement of total ammonia, a temperature of 15 °C, and the initial pH (pH i) of the sample (pH I = pH as measured on composite 100% sample at 15°C before any aeration of the test solutions). A pH stabilization technique must not be used if the concentration of un-ionized ammonia in a wastewater sample equals or exceeds 1.25 mg/L.

The pH must be measured and recorded at the beginning of the test (when fish are added to the wastewater effluent and control) and at each time interval required by each procedure, in all concentrations and the control (Sections 2.2.4, 2.3.2, and 2.4.3). Additional monitoring of pH during the first 8 hours of testing may be needed when using the pH stabilization procedure. For the remainder of the test, pH must be measured at each 24-h interval (at minimum) to track changes in pH and to ensure that the pH is maintained within test validity criteria (Section 2.1.3). More frequent monitoring of pH (e.g., twice daily) may be needed if the wastewater effluent sample has a low buffering capacity (low alkalinity), which may result in rapid changes in pH.

2.1.2 Chlorinated Wastewater Samples

Laboratories should measure total residual chlorine (TRC) concentrations in each wastewater effluent sample received (i.e., at the same time ammonia measurements are made). Total residual chlorine must be measured in the sample if fish display stressed or atypical behaviour at test initiation. If chlorine is present in the sample (TRC >0.1 mg/L), pH stabilization procedures should not be used, since trout mortality, stress or atypical behaviour will occur, regardless of pH drift. Testing laboratories should contact the wastewater facility to review their wastewater treatment operations (i.e., determine if the wastewater is chlorinated and then dechlorinated prior to discharge) and obtain historical data (i.e., determine typical total chlorine concentrations in the final discharge) because pH stabilization would not be necessary if the wastewater effluent samples contained a lethal level of total chlorine. Additional information on procedures to remove chlorine from wastewater effluent samples for investigative purposes is provided in Environment Canada (2008).

2.1.3 Test Validity Criteria

A test is considered invalid if any of the following occur:

1. the average pH in the pH stabilized 100% wastewater effluent test solution shifts more than ± 0.2 units from pH i;
2. the instantaneous pH in the pH stabilized 100% wastewater effluent test solution is greater than ± 0.3 units from pH i; or
3. if >10% of the fish (combined data if replicates are used) in the pH stabilized control die or exhibit atypical or stressed behaviour.

In the case of a multiple concentration test, an LC50 calculation must not include any exposure concentration where the pH criteria were not met.^Footnote 3

2.2 pH Stabilization Using the CO₂ Injection Technique

In the CO₂ Injection pH stabilization technique, the upward drift of pH is controlled by aerating the wastewater test solutions (including control) using a mixture of 15% CO₂, 21% oxygen (O₂), and 64% nitrogen (N₂) (referred to as CO₂ mix) blended with a source of lab air.

In addition to the standard equipment and facilities required to conduct EPS 1/RM/13, the following materials and equipment are required to use this pH stabilization technique:

• certified compressed cylinder containing a mixture of 15% CO₂, 21% O₂, and 64% N₂ with a CGA 590 outlet connector from a certified compressed gas supplier (e.g., Praxair, Air Liquide, BOC Gases)
• dual stage CP brass CGA 590 compressed gas regulator (e.g., Concoa, Fisherbrand, Restek, Cole-Parmer, VWR)
• six 150-mm flow meters with adjustable valves (approximate flow rate range 0 to 137 mL/min) (e.g., Cole Parmer, Gilmont, Scienceware)^Footnote 4
• six 150-mm flow meters with adjustable valve (approximate flow rate range 0 to 300 mL/min) (e.g., Cole Parmer, Gilmont, Scienceware)^Footnote 4
• plastic male pipe adapters 1/8 × 3/16" (0.32 × 0.48 cm) or equivalent to connect flow meters to plastic tubing
• flexible plastic air tubing
• Tygon® tubing (R-3606) or equivalent
• tapered “Y” shaped plastic air tubing connectors (e.g., VWR, Fisherbrand)
• assorted plastic tubing connectors
• two-way and four-way manifold valves (e.g., Cole Parmer)

Diagrams and photos showing a CO₂ injection technique test setup are provided in Figures 1 to 5.

Figure 1 Schematic Diagram of a Six-concentration Test Using the CO₂ Injection Technique

Each concentration has two flow meters. One flow meter is for the CO₂ mix and the other is for the lab air flow. One flow meter has a maximum flow rate of 300 mL/min and the other has a maximum flow rate of 137 mL/min. This allows for a range of 0.5% to 15% CO₂ for sample volumes between 20 and 40 L. The final percent CO₂ will determine which flow meter controls the CO₂ and lab air flows. Refer to the CO₂ Injection Tecnhique section for guidance on determining the final percent CO₂. The final flow to the aquaria must be 6.5 mL/min · L (i.e., for a 20 L sample volume, the final flow from the two flow meters must add up to 130 mL/min).

Figure 2 Front View of CO₂ Injection Technique - Control Board with Flow Meters

Each concentration has two flow meters with 137 mL/min and 300 mL/min maximum flow rates. The required percent CO₂ determines which flow meter controls the flow of CO₂ mix, and which flow meter controls the laboratory air.

Figure 3 Rear View of CO₂ Injection Technique - Control Board with Connections

Figure 4 Glass Aquaria Used for CO₂ Injection Technique

Figure 5 Plastic Pails Used for CO₂ Injection Technique

2.2.1 Apparatus for Delivering CO₂Mixture

The CO₂ is delivered to a test vessel from a compressed gas cylinder containing 15% CO₂, 21% O₂, and 64% N₂ mix via a gas cylinder regulator, flexible Tygon® air tubing through a 4-way gang valve, through a flow meter, combined with the normal lab airflow using a “Y” plastic connector and into the test solutions via an air stone. The normal lab air is delivered via flexible Tygon® R-3603 air tubing through a flow meter and combined with the CO₂ mix flow using the above-mentioned “Y” connector (Figures 1 and 3).

The cylinder containing the CO₂ mix should be securely attached near the exposure vessels by chaining to a wall or other stable structure. Never use oil or grease on regulator or cylinder fittings, as this could contaminate the pure gas mix, or create a fire hazard.

2.2.2 Estimating Percent CO₂ Mixture Required to Stabilize pH

The initial percent CO₂ mixture required to stabilize pH is estimated by measuring the initial pH (pH i) and alkalinity of the 100% test solution. In a multiple concentration (LC50) test, in order to determine the correct percent CO₂ mix at the start of the test: (i) initial pH (pH i) must be measured in each concentration, and (ii) alkalinity must be measured in the 100% wastewater effluent. Alkalinity in the remaining dilutions may be estimated, based on the known alkalinity values of the control/dilution water and the 100% wastewater effluent.

Once the initial pH and alkalinity have been determined, the CO₂ calibration table (Table 1) is used to estimate the percent CO₂ that is applied for a given initial pH and alkalinity to provide pH control. For example, a test solution with an alkalinity of 300 mg/L, as calcium carbonate (CaCO₃), with an initial pH of 7.1 will require a final flow of 5% CO₂  to maintain the initial pH. The CO₂ calibration table is for estimation and guidance purposes only.

Some adjustment to the percent CO₂ will be required if there is an upward or downward trend in pH after initiation of aeration with the CO₂ mixture. For example, the percent CO₂ mixture should be increased if there is an upward trend in the pH, while the percent CO₂ mixture should be lowered (decreased) if there is a downward trend in the pH.

The percent of CO₂ mixture required to stabilize pH depends on the chemical characteristics (i.e., alkalinity/buffering capacity) of the test solution. For example, wastewater effluent samples with a low buffering capacity require less CO₂ to control pH drift compared to those that have a greater buffering capacity.

2.2.3 Setting Flow Rates for CO₂ Mixture and Laboratory Air

In a multiple concentration (LC50) test setup (5 test concentrations, plus control), six flow meters with a 0 to 137 mL/min flow rate, and six flow meters with a 0 to 300 mL/min flow rate are required. All flow meters have adjustable valves. Each test concentration and control has one of each type of flow meter: one to control the flow of CO₂ mix, and the other to control the flow of lab air.

After the required percent CO₂ has been determined (Table 1), the flow rate of the CO₂ mix and lab air can be adjusted to achieve the required percent CO₂ using the adjustable valves on the flow meters. The test solution volume and required final percent CO₂ determines which flow meter is used to control the two flows of CO₂ mix and lab air.

The following equations are used to determine flow rates for the CO₂ mixture and lab air:^Footnote 5

(1) Combined flow to the test vessel (mL/min) = 6.5 mL/min·L × test volume (L)

(2) Flow rate of CO₂ mix = [Required % CO₂ from Table 1)] ÷ [% CO₂ in the mix (i.e., 15%)] × Combined flow to the test vessel (1)

(3) Flow of lab air = Combined flow to the test vessel (1) - Flow rate of CO₂ mix (2)

The CO₂ mix flow may be adjusted to maintain pH control anytime during the test.

2.2.4 Controlling pH Drift

Stabilization of pH commences immediately upon initiation of aeration at 6.5 ± 1 mL/min · L (see Section 2.1). Total aeration rates (CO₂ and lab air) must be 6.5 ± 1 mL/min · L throughout the test in all exposure concentrations, including the control (as per EPS 1/RM/13). Each test vessel is aerated through an air stone with lab air and CO₂ gas mix at a combined rate of 6.5 ± 1 mL/min · L at a percent CO₂ that maintains the average pH (for all effluent concentrations, excluding the control) within ± 0.2 pH units and the instantaneous pH within ± 0.3 pH units of pH i.^Footnote 6

Frequent pH measurements and appropriate adjustments to the flow of the CO₂ mix to stabilize pH must be conducted particularly during the first three hours of the test. Most adjustments to the flow of CO₂ mix occur within the first few hours from the start of aeration. Fewer adjustments (one to two times daily) will likely be required in the days after test initiation.

The pH must be measured and recorded immediately before any aeration (pH i), at t = 0 h (test start, when fish are introduced), and at t = 0.5, 1, 2, 3, 24, 48, 72 and 96 h in the control and all exposure concentrations. This will provide data to show that the pH has been maintained throughout the entire duration of the test. The pH must also be measured and recorded whenever there is an adjustment to the CO₂ flow. A subsequent pH reading must be taken 30 minutes or sooner after an adjustment, to ensure the pH is being maintained. The final pH is recorded if there is 100% rainbow trout mortality in a test concentration before the end of the 96-h test period.

If the test solution pH begins to decrease within the first 30 minutes of aeration, the percent CO₂ should be decreased in 0.5% increments until the pH is maintained within ± 0.2 pH units of the initial pH. If the test solution pH begins to increase in the first 30 minutes of aeration, increase the percent CO₂ in 0.5% increments until the pH is maintained within ± 0.2 pH units of the initial pH. Continue to make adjustments in 0.5% CO₂ increments until the pH is maintained within ± 0.2 pH units of the initial pH. The percent CO₂ delivered to each test vessel must be recorded.

In cases where the control/dilution water has a lower buffering capacity than the wastewater effluent test solution, it is unlikely that the amount of CO₂ needed to maintain pH in a sample would maintain a pH within ± 0.2 pH units of the laboratory control/dilution water initial pH. Therefore, in the single-concentration pH stabilization test, it is not required to add the same amount of CO₂ to both the 100% wastewater effluent sample and the control, since the purpose of this pH stabilization procedure is to control the pH drift of the effluent sample by replacing CO₂ that has been lost from the original test solution due to aeration during a rainbow trout acute lethality test.

Air line tubing must be inspected at least once a day to ensure continual delivery of CO₂ mixture and laboratory air to all test solutions.

2.3 pH Stabilization Using the Recycling Technique

The Recycling pH stabilization technique controls upwards pH drift by recycling CO₂ in a closed system (Elliott et al., 2003). A lid is placed securely on the test vessel and air, which contains CO₂, is re-circulated in the contained headspace with an air pump, thereby preventing loss of CO₂ to the atmosphere and maintaining the pH.

In addition to the standard equipment and facilities required to conduct EPS 1/RM/13, the following materials and equipment are required to use this pH stabilization technique:

• Tygon® Tubing (siphon tubes and air lines)
• aquarium aeration pump that generates 6.5 ± 1 mL/min · L and conforms with the lid (e.g., Elite 799 & 800, 115-volt aquarium aeration pump)
• 10 mL disposable pipettes
• recycle lids [with neoprene seals, O-rings, and elastics attached; lids are fabricated to fit test vessels used; can be fabricated and purchased from a plastic fabricator, e.g., Allwest Plastic Fabricators in Edmonton
• 250-mL Erlenmeyer catch flask used to prevent condensate from entering flow meters
• test vessels (aquaria or other containers such as pails)

Diagrams and photos showing a Recycle technique test setup are provided in Figures 6 and 7.

2.3.1 Recycling Technique Setup

To reduce headspace, the test container is filled to the very top with sample, without immersing the pump. The Recycle lid is then placed loosely on top of the test container. The air line tubing (#1) runs from the aeration pump to the catch flask as shown in Figure 6, and air line tubing (#2) then runs from the catch flask to the flow meter. The air line tubing (#3) proceeds from the flow meter outlet to a connector in the top of the Recycle lid. An air line (# 4) is attached to the bottom of the same connector and an air stone is attached to the end of this air line which is immersed in the sample. A siphoning tube is also attached to the Recycle lid for removing an aliquot of sample at each 24-h observation for physicochemical measurements. This is accomplished by attaching Tygon® tubing (#5) to a connector in the top of the Recycle lid and Tygon® tubing (#6) to the bottom of the same connector. A 10-mL pipette is attached to the end of this Tygon® tubing (#6) and immersed in the sample. After an aliquot has been removed, ensure that the end of the siphoning tube is stored above the sample level to avoid loss of sample. A tubing clamp can be used to seal the end of the siphoning tube as a precaution. Extra care must also be taken when collecting sub-samples for monitoring water quality parameters (e.g., pH, temperature), as a vacuum can form and result in a significant loss of sample from the test vessel.

The Recycle lid is secured and sealed to the top of the test container by fastening all O-rings and elastics around the plastic knob catches on the test vessel. The Recycling technique will only be successful in stabilizing pH if a tight seal is obtained.

Highly coloured, opaque, or foamy test solutions in the sealed test vessel used for the Recycling technique could make test observations difficult when inspecting fish for stress and mortality. Checks and removal of dead fish must be completed as quickly as possible to prevent pH drift.

For wastewater effluent samples with a high BOD, a decline in dissolved oxygen could be observed during an acute lethality test with rainbow trout when the pH is not stabilized. This dissolved oxygen decline could be accentuated if the Recycle technique were to be used to stabilize pH.

2.3.2 Controlling pH Drift

Stabilization of pH commences immediately upon initiation of aeration for 30 minutes at 6.5 ± 1 mL/min · L, before fish are added (see Section 2.1). To begin aeration, the pump is started and the flow meter setting is adjusted. All aeration rates must be 6.5 ± 1 mL/min · L throughout the test in all exposure concentrations, including the control (as per EPS 1/RM/13).

After the required aeration has been completed (i.e., minimum 30 minutes), the elastics from the sampling port on the Recycling lids for all concentrations are removed. The sampling port is opened, and fish are added in a quick and random manner. The sampling port is resealed before proceeding with the test.

Figure 6 Schematic Diagram of a Recycling Technique Test Unit

Figure 7 Glass Aquaria with Sampling Port, Air Pump, and Recycle Lid for Recycling Technique

The pH must be measured and recorded immediately before any aeration (pH i), at t = 0 h (test start, when fish are introduced) and then at 24, 48, 72, and 96 h in the control and all exposure concentrations. This will provide data to show that the pH has been maintained throughout the entire duration of the test. It is also recommended that pH be measured at t = 0.5, 1 and 2 h to ensure that the lid has been properly sealed. The pH must also be taken and recorded whenever the test container is opened (i.e., to remove dead fish). The final pH is recorded if there is 100% rainbow trout mortality in a test concentration before the end of the 96-h test period.

Any dead fish observed at each 24-h interval must be removed as quickly as possible. To remove dead fish from the test vessel, the elastics from the sampling port on the Recycle lid are removed and the dead fish are taken out. The elastics are then replaced to seal the test vessel. It is important that any opening of the test vessels be completed quickly, as a loss of CO₂ during this step may result in pH increases.

Visual checks must be made at least once per day to ensure that the air lines, pumps, and flow meters are working properly.

2.4 pH Stabilization Using the pH Controller Technique

The pH stabilization controller technique uses pure CO₂ (or a gas mix of 15% CO₂, 21% O₂ and 64% N₂) with separate lines for lab air addition. If pH drifts above a predetermined and programmed set point, the controller is activated and CO₂ is added to reduce pH. Once pH returns to the acceptable limit, the injection of CO₂ is automatically shut off.

In addition to the standard equipment and facilities required to conduct EPS 1/RM/13, the following materials and equipment are required to use this pH stabilization technique:

• solenoids (one for each exposure concentration) to control the flow of CO₂
• CO₂ pressure regulator and needle valve assembly
• certified compressed cylinder containing 100% CO₂ from a certified compressed gas supplier (e.g., Praxair) [note: gas mixture (15% CO₂, 21% O₂, and 64% N₂) from CO₂ Injection technique can also be used for pH Controller stabilization technique]
• pH controller [e.g., American Marine Inc. (cat.# CRT4) or equivalent; available from Fish Farm Supply, Elmira, Ontario]
• glass pipettes (1 mL)
• backflow valves (e.g., available from Hagen®)
• various fittings [1/2" (1.25 cm) black pipe (natural gas fittings) for LC50 setup; 1/2" × 90 deg; 1/2" 'T'; 1/2" × 2.5" nipples; 1/8" (0.32 cm) inner diameter Tygon® tubing to connect pipettes; 1/2" × 6" (152 cm) nipples]

A glass pipette is highly recommended for use in the delivery of the CO₂ gas because it provides better control of the amount of CO₂ gas that is delivered to the sample when the controller is activated. Diagrams and photos showing a pH Controller technique test setup are provided in Figures 8 to 13.

2.4.1 Regulator/Solenoid Assembly

Individual pressure regulators are connected to the gauge assembly (manifold) (Figures 8 and 9). The CO₂ gas regulator is connected to a CO₂ cylinder. Never use oil or grease on regulator or cylinder fittings, as this could contaminate the pure gas mix, or create a fire hazard.

The manifold is connected to the regulator on the CO₂cylinder using high-pressure polypropylene tubing (1/4" [0.64cm] outer diameter). All needle valves on the solenoids must be turned to the off position. The locking hex cap from the regulator on the solenoid assembly is removed to expose the adjustment screw (hex wrench or Allen key may be required) and the screw is turned counterclockwise until there is no more resistance.

The valve on the CO₂ cylinder is opened and pressure is adjusted to approximately 40 psi. The working pressure on the solenoid is adjusted (using the hex wrench or Allen key) to approximately 20 psi. Connections should be tested for leaks using a dilute dish detergent (any bubble formation suggests there may be leaks and the system should be rechecked and sealed as required).

An appropriate length of silicone airline (1/4" outer diameter) is connected to the needle valve and attached to the pipette/back-flow preventer (Figure 13).

2.4.2 pH Controller

The pH Controller (see Figure 12) must be calibrated daily using certified pH standards. The tolerance of the pH Controller (i.e., the sensitivity of pH control) must be set before test initiation (± 0.2 pH units). The CO₂ tubing must be removed from the exposure solution during calibration. Meter calibration must be completed rapidly to prevent pH drift from occurring. Instructions for calibration and maintenance should be provided by the manufacturer and reviewed before test initiation.

The pH probe from one controller is placed into a single test solution for the duration of the test (the probe can be temporarily removed for calibration). The probe should be secured 3-5 cm below the surface of the test solution. The CO₂ delivery pipette should be directly beneath the pH probe, and tied to the probe conductor. This is important for accurate pH control. Back siphoning into the CO₂ line could occur, but this can be prevented by using a spring-loaded (stainless steel) back-flow check valve (Figure 13). Durable pH probes should be used to reduce the risk of leaks of potassium chloride (KCl) solution from the probe into the exposure solution.

Figure 8 Schematic Diagram of a Six-concentration Test Using pH Controller Technique

Figure 9 Solenoid with Regulator and Needle Valve Assembly for pH Controller Technique

Figure 10 Test Setup for pH Controller Technique

Figure 11 Overview of Setup for LC50 Test Using pH Controller Technique

Figure 12 Example of pH Controller Unit

Figure 13 Supply Line for CO₂Showing Backflow Preventer Valve

2.4.3 Controlling pH Drift

Stabilization of pH commences upon initiation of aeration for 30 minutes at 6.5 ± 1 mL/min · L, before fish are added (see Section 2.1). When aeration is started, the main valve on the CO₂ cylinder is opened to approximately 40 psi. Pressure readings at the solenoid regulator gauge should be approximately 20 psi.

Aeration rates of laboratory air must be at 6.5 ± 1 mL/min · L throughout the test in all exposure concentrations, including the control (as per EPS 1/RM/13). Each test vessel is aerated through an air stone with lab air at 6.5 ± 1 mL/min · L. The addition of CO₂ will slightly increase the aeration rate each time the pH controller cycles on or off, in order to maintain the mean pH in the 100% test solution within ± 0.2 pH units and the instantaneous pH within ± 0.3 pH units of the initial pH. The increase in aeration rate is considered insignificant, since it only occurs periodically to control upwards pH drift and should still be within the allowable limits.

Taking frequent pH measurements and making appropriate adjustments to the flow of CO₂ is critical to stabilizing pH, particularly during the first few hours of the test. The pH value on the controllers must be closely monitored to ensure proper operation of the solenoid. It is important for the controller to cycle on and off to control the flow of CO₂. If cycling does not occur within two to five minutes of operation, and the solenoid remains open (powered), then CO₂ flow should be gradually increased using the needle valve until the required pH value is reached and the solenoid closes.

The pH must be measured and recorded immediately before any aeration (pH i), at t = 0 h (test start, when fish are introduced) and then at 24, 48, 72 and 96 h in the control and all exposure concentrations. This will provide data to show that the pH has been maintained throughout the duration of the test. The pH must also be measured and recorded whenever there is a manual adjustment to the CO₂ flow. A subsequent pH reading must be taken 30 minutes or sooner after an adjustment, to ensure the pH is being maintained. The final pH is recorded if there is 100% rainbow trout mortality in a test concentration before the end of the 96-h test period.

Visual checks must be made once per day to ensure that the pH Controllers and air lines are working properly.