Facilities
(2) Facilities(i) General. (A) Facilities needed to perform this test include: a growth chamber or a controlled environment room that can hold the test containers and will maintain the air temperature, lighting intensity and photoperiod specified in this test guideline; apparatus for culturing and enumerating algae; a source of distilled and/or deionized water; and apparatus for carrying out analyses of the test chemical. (B) Disposal facilities should be adequate to accommodate spent glassware, algae and test solutions at the end of the test and any bench covering, lab clothing, or other contaminated materials. (ii) Test containers. Erlenmeyer flasks should be used for test containers. The flasks may be of any volume between 125 and 500 ml as long as the same size is used throughout a test and the test solution volume does not exceed 50 percent of the flask volume. (iii) Cleaning and sterilization. New test containers may contain substances which inhibit growth of algae. They shall therefore be cleaned thoroughly and used several times to culture algae before being used in toxicity testing. All glassware used in algal culturing or testing shall be cleaned and sterilized prior to use according to standard good laboratory practices. (iv) Conditioning. Test containers should be conditioned by a rinse with the appropriate test solutions prior to the start of the test. Decant and add fresh test solutions after an appropriate conditioning period for the test chemical. (v) Nutrient medium. (A) Formulation and sterilization of nutrient medium used for algal culture and preparation of test solutions should conform to those currently recommended by the U.S. EPA for freshwater and marine algal bioassays. No chelating agents are to be included in the nutrient medium used for test solution preparation. Nutrient medium should be freshly prepared for algal testing and may be dispensed in appropriate volumes in test containers and sterilized by autoclaving or filtration. The pH of the nutrient medium shall be 7.5 (0.1) for Selenastrum and 8.1 (0.1) for Skeletonema at the start of the test and may be adjusted prior to test chemical addition with 0.1N NaOH or HC1. (B) Dilution water used for preparation of nutrient medium and test solutions should be filtered, deionized or glass distilled. Saltwater for marine algal nutrient medium and test solutions should be prepared by adding a commercial, synthetic, sea salt formulation or a modified synthetic seawater formulation to distilled/deionized water to a concentration of 30 parts per thousand. (vi) Carriers. Nutrient medium shall be used in making stock solutions of the test chemical. If a carrier other than nutrient medium is absolutely necessary to dissolve the chemical, the volume used shall not exceed the minimum volume necessary to dissolve or suspend the chemical in the test solution. (3) Test parameters. (i) The test temperature shall be 24 C for Selenastrum and 20 C for Skeletonema. Excursions from the test temperature shall be no greater than 2 C. Temperature should be recorded hourly during the test. (ii) Test chambers containing Selenastrum shall be illuminated continuously and those containing Skeletonema shall be provided a 14-hour light and 10-hour dark photoperiod with a 30 minute transition period under fluorescent lamps providing 300 25 uEin/m 2 sec (approximately 400 ft-c) measured adjacent to the test chambers at the level of test solution. (iii) Stock algal cultures should be shaken twice daily by hand. Test containers shall be placed on a rotary shaking apparatus and oscillated at approximately 100 cycles/minute for Selenastrum and at approximately 60 cycles/minute for Skeletonema during the test. The rate of oscillation should be determined at least once daily during testing. (iv) The pH of nutrient medium in which algae are subcultured shall be 7.5 (0.1) for Selenastrum and 8.1 (0.1) for Skeletonema, and is not adjusted after the addition of the algae. The pH of all test solutions shall be measured at the beginning and end of the test. (v) Light intensity shall be monitored at least daily during the test at the level of the test solution.