§ 252.403. Essential quality control requirements—toxicity testing.
(a) In addition to the requirements of § 252.401 (relating to basic requirements), an environmental laboratory that measures the toxicity or bioaccumulation of contaminants, including testing of effluents, receiving waters, sediments, elutriates, leachates and soils shall comply with this section.
(b) When the method selected by an environmental laboratory in accordance with § 252.307 (relating to methodology) contains more stringent requirements than the requirements of this section, the environmental laboratory shall follow the more stringent requirements contained in the method.
(c) An environmental laboratory that measures toxicity or bioaccumulation of contaminants shall comply with Chapter 16 (relating to water quality toxics management strategy—statement of policy) regarding counting of neonates, algae cells and weighing of fish for selected endpoints.
(d) Negative control requirements are as follows:
(1) In addition to the negative controls specified by the method, permit or regulation, additional negative controls shall be included when sample adjustments (for example, pH adjustments or dechlorination) or solvent carriers are used in the test.
(2) The results of the negative controls shall be compared to the acceptance criteria published in the method. When there are no established acceptance criteria for the negative control in the method, the environmental laboratory shall establish internal criteria and document the method used to establish the acceptance limits.
(3) The test acceptability criteria for negative controls as specified in the method must be achieved for both the reference toxicant and the environmental sample toxicity test.
(e) The requirements for reference toxicants are as follows:
(1) The environmental laboratory shall demonstrate the ability to obtain consistent results with reference toxicants before performing toxicity tests on environmental samples.
(i) Intralaboratory precision shall be determined by performing a minimum of five acceptable reference toxicant tests for each method and species using different batches of organisms and negative controls (water, sediment or soil) before performing testing or analysis on environmental samples.
(ii) An environmental laboratory shall maintain control charts for the control performance and reference toxicant statistical endpoint (such as NOEC or ECp) and evaluate the intralaboratory variability with a specific reference toxicant for each method.
(iii) The results of the toxicant test shall be compared to the acceptance criteria published in the method. When there are no established acceptance criteria for the toxicant test in the method, the environmental laboratory shall establish internal criteria and document the method used to establish the acceptance limits.
(2) The following minimum frequency of reference toxicant testing shall be met:
(i) Each batch of test organisms obtained from an outside source, field collection or from laboratory spawning of field-collected species not amenable to routine laboratory culture shall be evaluated with a reference toxicant test of the same type as the environmental toxicity test within 7 days preceding the test or concurrently with the test.
(ii) Test organisms obtained from in-house laboratory cultures shall be tested with reference toxicant tests at least once each month for each method.
(iii) If a species produced by in-house laboratory cultures is used less than once per month, a reference toxicant test of the same type shall be performed with each environmental toxicity test.
(iv) When methods and species commonly used in the laboratory are only tested on a seasonal basis, reference toxicant tests shall be conducted each month the method is in use.
(3) Ongoing environmental laboratory performance shall be documented by maintaining laboratory quality control charts that meet the following requirements:
(i) For endpoints that are point estimates (ICp, ECp), control charts shall be constructed by plotting the cumulative geometric mean and the limits that consist of the upper and lower 95% confidence limits (+2 standard deviations).
(ii) For endpoints from hypothesis tests (NOEC, NOAEC), control charts shall be constructed by plotting the values directly and the control limits shall consist of one concentration interval above and below the concentration representing central tendency or the mode.
(iii) After 20 data points are collected for a method and species, the control charts shall be maintained by using only the most recent 20 data points.
(iv) Test results that fall outside of control chart limits at a frequency of 5% or less shall be retested and confirmed before reporting and all results shall be documented in the report of the testing and analysis.
(v) The endpoint shall be compared to the acceptance criteria published in the method.
(vi) When there are no established acceptance criteria for the endpoint in the method, the environmental laboratory shall establish internal criteria and document the method used to establish the acceptance limits.
(vii) If the reference toxicant fails to meet acceptance criteria, the results of environmental toxicity tests conducted during the affected period shall be examined for defects and the test repeated using a different batch of organisms or the results shall be reported with appropriate data qualifiers.
(4) Reference toxicant tests conducted for a method and species must use the same reference toxicant, test concentrations, dilution water and data analysis method as the environmental toxicity tests for which the precision is being evaluated unless otherwise specified in the method.
(5) The test duration, dilution or control water, feeding, organism age, age range and density, test volumes, renewal frequency, water quality measurements, number of test concentrations, replicates and organisms per replicate must be the same as the environmental toxicity test. A dilution factor of greater than 0.5 shall be used for both acute and chronic tests.
(f) Sensitivity requirements are as follows:
(1) If the Dunnett’s procedure or hypothesis test (NOEC, NOAEC) is used, the statistical minimum significant difference (SMSD) by species shall be calculated according to the formula specified by the method and reported with the test results. The SMSD must be estimated for nonnormal distribution or heterogeneous variances, or both.
(2) Confidence intervals for point estimates (LCp, ICp or ECp) shall be reported as a measure of the precision around the point estimate value.
(g) When required, the data shall be plotted in the form of a curve relating the dose of the chemical or concentration of sample to cumulative percentage of test organisms demonstrating a response, such as death.
(h) At least once every 30 days, an environmental laboratory shall verify and document that the reagent grade water meets the following criteria:
(1) Conductivity must be less than 0.1 µmhos/cm or resistance greater than 17 megohms at 25°C.
(2) pH must be between 5.5 to 7.5.
(3) Total residual chlorine must be nondetectable.
(i) Reagent water used for culturing and testing shall be analyzed for toxic metals and organics whenever the minimum acceptability criteria for control survival, growth or reproduction are not met and no other cause can be identified.
(j) An environmental laboratory shall demonstrate that any analyte at a measured concentration or the reported detection limit does not exceed 1/10 the expected chronic value for the most sensitive species tested or cultured.
(k) Air used for aeration of test solutions, dilution waters and cultures must be free of oil and fumes.
(l) The requirements for test organisms are as follows:
(1) An environmental laboratory shall positively identify test organisms to species on an annual basis. The taxonomic reference (citation and pages) and the names of the taxonomic experts shall be documented. When organisms are obtained from an outside source, an environmental laboratory shall obtain the information from the supplier.
(i) Organisms used for a test must be from the same source. When available, certified seeds shall be used for soil tests.
(ii) Organisms used in tests or as brood stock to produce neonate test organisms must appear healthy, show no signs of stress or disease and exhibit survival of greater than 90% during the 24-hour period immediately preceding use in tests.
(iii) An environmental laboratory shall document the health and culturing conditions of all organisms used for testing. The documentation must include culture conditions and observations of any stress, disease or mortality.
(iv) When organisms are obtained from an outside source, the laboratory shall obtain written documentation of the water quality parameters and biological observations for each lot of organisms received.
(v) An environmental laboratory shall record the water quality parameters and the biological observations when the organisms arrive at the environmental laboratory.
(vi) Supporting information such as hatch dates and times, times of brood releases and metrics (for example, chironomid head capsule width) shall be documented.
(vii) Organisms obtained from an outside source may not be from different batches.
(viii) The control population of Ceriodaphnia in chronic effluent or receiving water tests may not contain more than 10% males.
(ix) Test soils and sediments must be within the geochemical tolerance range of the test organism.
(2) The requirements for feeding of test organisms are as follows:
(i) For each new batch of laboratory-prepared food or lot of commercial food used by the environmental laboratory, the performance of organisms fed with the new food shall be compared with the performance of organisms fed with a food of known quality. The suitability of food used for culturing shall be determined using a measure that evaluates the effect of food quality on survival and growth or reproduction of each of the relevant test species.
(ii) Foods used only in chronic toxicity tests shall be evaluated using the reference toxicant employed in the environmental laboratory quality assurance program, and shall be compared with results of previous tests using a food of known quality.
(iii) In the case of algae, rotifers or other cultured foods, which are collected as a continuous batch, the quality of the food shall be assessed as described in subparagraphs (i) and (ii) each time new nutrient stocks are prepared, a new starter culture is employed or when a significant change in culture conditions occurs.
(iv) The environmental laboratory shall have written procedures for the statistical evaluation of food acceptability.
(v) Food used to culture organisms used in bioaccumulation tests shall be analyzed for the compounds to be measured in the bioaccumulation tests.
(m) Equipment requirements are as follows:
(1) If closed incubators are used, culturing and testing of organisms shall be separated to avoid loss of cultures due to cross-contamination.
(2) Temperature control equipment must be adequate to maintain the required test temperature. The average daily temperature of the test solutions shall be maintained within 1°C of the selected test temperature for the duration of the test. Temperature measurements shall be made at least once per 24-hour period. The test temperature for continuous-flow toxicity tests shall be monitored and recorded continuously.
(3) The test chambers used in a test must be identical.
(4) Materials used for test chambers and any material coming in contact with test samples, solutions, control water, sediment, soil or food must be nontoxic and cleaned according to the method. Materials may not add to nor reduce sample toxicity.
(5) Light intensity shall be maintained as specified in the method. Measurements shall be made and recorded at least once per 12 months.
(6) The photoperiod shall be maintained as specified in the method and be documented at least once every 90 days.
(7) For algal and plant tests, the light intensity shall be measured and recorded at the start of each test.
(n) The requirements for sample holding times and conditions are as follows:
(1) The sample holding time may not exceed 36 hours.
(2) The last use of the sample in renewal tests may not exceed 72 hours unless specifically approved by the Department.
(3) Samples shall be chilled to 4°C during or immediately after collection and held at that temperature until time of analysis.
(o) Chronic tests must have a minimum of four replicates per treatment.
(p) The requirements for testing conditions are as follows:
(1) Dissolved oxygen and pH in aquatic tests must be within acceptable ranges published in the method. When there are no established acceptance criteria in the method, the environmental laboratory shall establish internal criteria and document the method used to establish the acceptance limits.
(2) During aquatic chronic testing, dissolved oxygen and pH shall be measured daily in at least one replicate of each concentration.
(3) In static-renewal tests, dissolved oxygen shall be measured at both the beginning and end of each 24-hour exposure period.
(4) The pH shall be measured at the end of each exposure period after organism transfer.
(5) Minimal aeration may be provided to tests only if acceptable dissolved oxygen concentrations cannot be otherwise maintained or if specified by the method.
(q) Records of all equipment, reference materials, reagents and supplies shall be maintained in accordance with § 252.306 (relating to equipment supplies and reference materials).
Authority The provisions of this § 252.403 amended under 27 Pa.C.S. § § 4103(a), 4104 and 4105; and section 1920-A of The Administrative Code of 1929 (71 P. S. § 510-20).
Source The provisions of this § 252.403 amended April 9, 2010, effective April 10, 2010, 40 Pa.B. 1898. Immediately preceding text appears at serial pages (317279) to (317284).
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