Toxicity Test for Antisapstain Regulation

Toxicology / bioassay
Sparcode: 0459X542
Revision Date: February 8, 2001

96HRLT50 Fish Bioassay with Hardness and pH Adjustments

(Note: This method is applicable only for the Environmental Management Act Antisapstain Chemical Waste Control Regulation, B.C. Reg. 300/90).

0459 - Bio. LT50 Fish

Freshwater bioassay with less than 10 parts per thousand (ppt) salinity in sample and control

Analytical Method
X542 – Rainbow/96hr/pH, hardness adj. (% surv)

Toxicology. Oncorhynchus mykiss (rainbow trout)

Method Summary
Under this test, rainbow trout are exposed to a hardness and pH-adjusted undiluted effluent sample for a period of 96 hours.

Background receiving water samples should be taken at points as described in Appendix 1-A and analyzed for hardness and pH.

Effluent samples, that have a lower hardness than that of the receiving water to which the effluent is discharged, are adjusted in the laboratory prior to testing, to equal either:

  • the current measured hardness and pH in the receiving water (see Appendix 1-B, Method 1); or
  • the hardness and pH of the receiving water as determined from records (see Appendix 1-B, Method 2).

The procedure for adjusting hardness and pH is described in Appendix 1-C.

Effluent samples that have a higher hardness than the receiving water to which the effluent is discharged, are not adjusted for hardness prior to testing.

A control bioassay of hardness and pH-adjusted laboratory control water is run concurrently with the sample bioassay.

There is no adjustment of the effluent sample or the laboratory control water based on the salinity of the receiving waters.

Precipitation runoff from lumber treatment or lumber storage areas.

Interferences and Precautions
The following sample properties may affect the test results: extreme volatility, instability, excessive oxygen demand, extreme temperatures, extremes in pH, or extreme concentrations of suspended solids. Control water exhibiting extremes of water temperature, hardness, or containing suspended solids, toxic chemicals or metals, may cause problems. Precautions must be taken to ensure proper handling of test organisms including proper acclimatization, freedom from disease and previous prophylactic treatment.

Sample Handling
For 96HRLT50, two clean 20 litre plastic containers. Expel all air pockets. Ship with ice packs. For hardness and pH determination, one clean 0.5 L container for each receiving water sample.

No preservation required. Store in dark at 4° C. Maximum holding time (MHT) – 5 days.

Greater than 10% mortality in either of the controls renders the bioassay invalid. The test is also invalid if greater than 10% of the fish exhibit atypical / stressed behaviour in either of the controls.

Quality Control
Reference toxicant warning chart data on test fish. Routine chemistry of holding and dilution water. Stock history of test fish.

Environment Canada, Biological Test Method: Acute Lethality Test Using Rainbow Trout. Report EPS 1/RM/9 July 1990 and EPS 1/RM/13 July 1990

British Columbia Environmental Laboratory Manual For the Analysis of Water, Wastewater, Sediment and Biological Material, 1994. Laboratory Services, Environmental Protection Department, Ministry of Environment, Province of British Columbia

British Columbia Field Sampling Manual, Clark, M.J.R. (editor), 1996. Laboratory and Systems Management, Environmental Protection Department, Ministry of Environment, Lands and Parks, Victoria, B. C., Canada

Appendix 1

A – Sampling for Determining Receiving Water Hardness and pH

In the absence of site-specific monitoring point(s) for determining receiving water hardness and pH, the sampling points should be located approximately 100 meters upstream of the discharge point. If there is a continuous upstream-downstream gradient in flow, then only the upstream hardness and pH needs to be determined. If there is no gradient to flow or if the flows are tidally reversed, then average values should be determined, based on samples collected at points 100 meters from the discharge point and along a line parallel to the shoreline.

B – Analytical Methods for Determining Hardness and pH for Bioassay Purposes

Method 1:

For immediate determination of hardness and pH, use procedures described in "Standard Methods for the Examination of Water and Wastewater" published by the American Public Health Association, 1995 (APHA). EDTA titration can be used to rapidly determine hardness. Alternately, separate determination of calcium and magnesium concentrations using procedures such as atomic absorption or ICP can be used to calculate hardness.

Method 2:

Records may be used to determine the hardness and pH in the receiving water if the hardness of site-specific receiving waters have been assessed no less than four times over a period of at least one year, and represent a variety of conditions.

The variety of conditions for assessing the hardness of receiving environments would include high and low flows in rivers, and flooding and ebbing tidal conditions in marine and estuarine environments.

When records from current or prior assessments of the receiving water are used to determine the hardness and pH of the receiving water, the lowest seasonal recorded values should be used in adjusting the sample and control in the toxicity test.

C - Procedure for Adjusting Hardness and pH

The hardness of the sample and control (consisting of laboratory control water) are adjusted without dilution to within ± 10% of that of the receiving water to a maximum of 300 ± 30 mg/L (as Ca CO3) using reagent-grade salts (APHA, 1995).

The salts should be thoroughly mixed in the sample or laboratory control water using a clean glass stirring-rod until dissolved. Alternatively, the salts can first be dissolved in a small amount of sample or laboratory control water on a stir plate prior to adding to the test container.

The pH is checked in the hardness-adjusted sample and control water (after the salts have been added), and adjusted using solutions of dilute acid or base.

Allow the sample to come to equilibrium during the 30-minute sample pre-aeration period and confirm the hardness and pH of the adjusted sample and control water prior to adding the fish.

Table 1 shows the amounts of salts required to increase the hardness by approximately 10 mg/L in one litre of test solution.

Table 1. Amount of reagent-grade salts to add to 1 L of water to increase hardness by 10 mg/L.


For example, if the sample hardness is 12 mg/L and the hardness downstream of the discharge is 17 mg/L, the hardness of the sample needs to be increased by 5 mg/L. Since the values in Table 1 are for 10 mg/L, they need to be multiplied by 0.5 to achieve the correct amount of salts to add per litre. This factor is obtained by the following equation:

Hardness adjustment factor (HAF) = (receiving environment hardness - sample hardness) ÷10

In our example, HAF = (17 - 12) ÷ 10 = 0.5. Alternatively if the receiving environment hardness was 25 mg/L and the sample was 12 mg/L, the HAF would be (25 - 12) ÷ 10 = 1.3.

Multiplying the values in Table 1 by the HAF (0.5) gives the following amounts of chemicals to be added per litre: NaHCO3, 6 mg; CaSO42H2O, 3.75 mg; MgSO4, 3.75 mg; and KCl, 0.25 mg. These values are then multiplied by the test volume to determine the total amount to add to the sample. For example, if the test volume was 15 L, the values would be multiplied by 15. Thus, for our example, the total amount of each salt added to a 15 L exposure volume would be: NaHCO3: 90 mg; CaSO4 2H2O: 56.25 mg; MgSO4: 56.25 mg; and KCl: 3.75 mg.

Signed by: Ron Driedger, Director of Pollution Prevention & Remediation Branch on February 20, 2001.