The materials and methods used in this thesis research are provided in this section. Acute and chronic toxicity testing was conducted on BC resident species of fish, invertebrates and algae. Literature data were also gathered in support of the data collected from toxicity testing on BC species. All relevant data were used to improve the existing freshwater aquatic life guideline for manganese contained in the BC Environment document Approved And Working Criteria For Water Quality (BCMELP, 1995).

3.1 B.C. PROTOCOL

The BC Ministry of Environment, Lands and Parks has developed procedures for deriving water quality criteria in British Columbia. These procedures are described in the document Derivation Of Water Quality Criteria To Protect Aquatic Life In British Columbia (September, 1995 Draft). This document outlines the minimum requirements that need to be met for data to be used in deriving water quality criteria and the minimum numbers of tests for each class of organisms (fish, invertebrates and plants) required to derive full and/or interim guidelines for the protection of aquatic life. The reader is referred to Appendix B for further details. The BC Environment procedures (BC Protocol) are similar to the Canadian Council of Ministers of Environment procedures detailed in the document A Protocol For The Derivation Of Water Quality Guidelines For The Protection Of Aquatic Life (CCME, 1987) presented in Appendix C.

Applying the BC Protocol will allow the objectives of this thesis research to be fulfilled by addressing the following key components:

1. Review of published and unpublished literature data
2. Determination of data requirements where literature sources do not provide sufficient data for water quality derivation purposes.
3. Use of new toxicity data in the derivation of water quality criteria protective of aquatic life.

The need for additional data on British Columbia species to supplement the data available in the literature was identified during the establishment of the 1995 freshwater aquatic life guideline for manganese. A toxicity testing program was therefore undertaken using species native to BC The aquatic toxicity testing procedures and methodologies were based on standard Environment Canada protocols, which also incorporated procedures adopted from organizations such as ASTM. Additional details pertaining to the testing methodologies utilized are provided in Appendix A. The species included in the suite of toxicity tests along with the toxicity endpoints measured are presented in Table 3.1.

Note: LC₅₀ - interpolated concentration at which 50% lethality occurs in test organisms versus control group

IC₅₀ - interpolated concentration at which 50% inhibition of toxicity endpoint (e.g. light production, plant mass) occurs in test organisms versus control group

Details regarding the sources from which test organisms were obtained, summaries and references for the toxicity tests, and quality assurance/quality control information including the acceptable ranges for water quality criteria (pH, DO, temp.) and the statistical methods applied to each test are presented in Appendix A.

Manganese chloride (MnCl₂) was chosen as the chemical form to prepare dissolved manganese test solutions for use in the toxicity testing program. A stock solution of 10 000 mg MnCl₂ dissolved in 1 litre of de-ionized water was prepared as required and test concentrations were prepared by placing pre-measured volumes of stock solution in a volumetric flask and filling with de-ionized water to achieve the desired concentration. Test concentrations varied based on the type of test, the organisms under study and observations/test results noted during the testing programs.

Existing information on manganese and other similar metals such as copper and zinc suggested a relationship between aquatic toxicity and water hardness. In order to further explore this relationship, three nominal water hardness values were chosen for evaluation in several of the toxicity tests, specifically 25 mg/L CaCO₃, 100 mg/L CaCO₃ and 250 mg/L CaCO₃. A ground water well with a water hardness of 100 mg/L was used as the water source for the freshwater testing program. The 25 mg/L softer water was prepared by diluting the well water with de-ionized water while the 250 mg/L hard water was prepared by reconstituting well water. All toxicity tests were conducted using a hardness value of 100 mg/L CaCO₃, with a portion of the tests conducted at all three water hardness values.

3.2 APPLICATION OF BC PROTOCOL

Classification of toxicity testing data as primary, secondary or unacceptable is required under the BC Protocol. The requirements for primary data include preferred partial or full life cycle exposure endpoints such as embryonic development effects, hatching or germination success, survival of juvenile stages, and growth, reproduction and survival of adults. For secondary data, the requirements include those for primary data as well as pathological, behavioural and physiological effects. The detailed requirements for primary and secondary data are described in Table 3.1 of Appendix B. Unacceptable data are those that do not meet the requirements of either primary or secondary data.

The studies under consideration for use in guideline derivation are also classified as acute or chronic and the types of organisms used in the tests are assessed. Minimum numbers of acute and chronic tests on species of fish, invertebrates and aquatic plants are required for full and interim guideline derivation. Details are provided in Appendices B and C, and in Section 4.1.2.

Data that is acceptable for use in guideline derivation is then reviewed to determine the concentrations at which adverse effects were observed. The lowest observed effect concentration or LOEC and the no observed effect concentration or NOEC are reviewed as are statistically derived effects concentrations such as LC₅₀s and IC₂₅s. These concentrations are compared with acceptable data for all organisms to determine the lowest concentrations from acute and chronic studies, which should be indicative of the more sensitive organisms under acute and chronic exposure conditions. As the objective of guideline derivation is the protection of aquatic life, organisms that are less tolerant of a substance in freshwater require weigh more heavily in the establishment of a guideline. Once a minimum value (or values) has been established, a safety factor is applied to compensate for uncertainty associated with the data set. The BC Protocol suggests a typical range of 0.1 to 0.5 (Section 4.1.1, Appendix B) depending on the quality of data and degree to which the toxicity of the particular substance is understood. The final acute and/or chronic guidelines with the safety factor applied are compared to the available data to ensure there is sufficient protection of sensitive species.