ANALYTICAL METHODS--Discontinuation of the National Water Quality Laboratory determinations for "total" nitrite, "total" nitrite plus nitrate, "total" ammonia,and "total" orthophosphate (using the four-channel analyzer) In Reply Refer To: December 2, 1992 Mail Stop 412 OFFICE OF WATER QUALITY TECHNICAL MEMORANDUM 93.04 Subject: ANALYTICAL METHODS--Discontinuation of the National Water Quality Laboratory determinations for "total" nitrite, "total" nitrite plus nitrate, "total" ammonia, and "total" orthophosphate (using the four-channel analyzer) SYNOPSIS The purpose of the memorandum is to inform Division personnel that determinations of "total" nitrite, "total" nitrite plus nitrate, "total" ammonia, and "total" orthophosphate in both the standard and low-level concentration ranges are being removed from the National Water Quality Laboratory (NWQL) catalog. Effective immediately, project personnel are instructed to discontinue requests for the laboratory codes (LC) listed below: Nutrient species and bottle designation Laboratory codes: Routine Low level ____________________________________________________________ Ammonia, Total.....................RC LC0123 LC0836 Nitrite, Total.....................RC LC0302 LC0840 Nitrite plus nitrate, Total........RC LC0304 LC0839 Orthophosphate, Total..............RC LC0297 LC0838 ____________________________________________________________ These laboratory analytical codes will be deleted from the laboratory catalog and the Schedules, Parameters, and Network Files System on January 1, 1993. Project personnel must check the laboratory schedules they are using to determine if the laboratory codes listed above are included. Deletion of these laboratory codes follows a technical examination of the method used to produce data for nutrient species on unfiltered and filtered samples, and from a statistical evaluation of the data produced by NWQL during 1989. The conclusion reached from evaluations of the method and data is: no valid basis exists for distinguishing between unfiltered and filtered determinations of nutrient species using the four-channel method. Moreover, the continuous-flow analyzer used in the four- channel method is unsuitable for unfiltered samples. Division personnel are advised of the following changes related to deletion of the laboratory codes listed above: 1. After January 1, 1993, no analysis will be run for nitrite, nitrite plus nitrate, ammonia, or orthophosphate on RC (Raw, Chilled) samples. The only RC samples to be analyzed will be those that request LC1686 for total phosphorus and (or) LC1688 for total Kjeldahl nitrogen (ammonia plus organic nitrogen). 2. All samples submitted to the laboratory for determination of nutrient species using the four-channel method must be designated "FC" (Filtered, Chilled) and, therefore, must be filtered in the field. 3. Effective immediately, project personnel may not request reruns on nutrient determinations using the four-channel method based on a criterion that concentrations reported in the RC (unfiltered) sample must equal or exceed those on the FC (filtered) sample. PROBLEM Determination of "total" (unfiltered) and "dissolved" (filtered) nitrite, nitrite plus nitrate, ammonia, and orthophosphate are requested routinely on identical or replicate samples. Concentrations of these nutrient species are determined simultaneously using a four-channel, continuous flow, colorimetric analyzer. No digestion is performed on unfiltered (RC) samples prior to analytical determinations; analytical procedures are identical for "dissolved" and "total" determinations. Therefore, no analytical basis exists for the distinction between analyte concentrations in the "dissolved" and "total" fractions. Moreover, unfiltered samples with large concentrations of solids can foul the four-channel analyzer on which these determinations are performed. Therefore, these samples may be laboratory filtered at the analyst's discretion, although the results are reported as a "total" concentration. Records of laboratory filtrations have not been kept. The problems inherent in using the four-channel method for nutrient determinations on unfiltered samples have been summarized by C.J. Patton of the Methods Research and Development Program, NWQL: 1. Colorimetric analysis of turbid solutions is contrary to accepted analytical practice. Particulates in reagent-treated samples scatter light, resulting in increased absorbance that is indistinguishable from absorbance caused by the colorimetric reaction. The consequence is to bias the analysis for unfiltered samples with appreciable particulate material toward higher concentrations than for filtered samples. 2. Rigorous chemical digestion is required to measure "total" nutrient concentrations in environmental samples. Unfiltered (RC) samples are not digested prior to analytical determinations by the four-channel method. 3. The method was not designed for turbid samples, nor does the equipment function optimally when samples with high particulate concentrations are introduced. The continuous-flow analyzer used in the four-channel method has conduits with internal diameters of 1 millimeter or less. Particulates in the "RC" samples can coat or partially block the mixing coils and flow cells of the colorimetric continuous-flow analyzers. This causes analytical problems that range from an abrupt shift in baseline absorbance to total malfunction. HISTORICAL BACKGROUND The rationale that originated the analysis for "total" concentrations of these four nutrient species in unfiltered samples is not well documented. The practice arose, in part, from a confusion embedded within our terminology. An unfiltered sample yields, by definition, a "total" analysis (that is, an analysis of the raw sample). Therefore, the concentrations analyzed in unfiltered nutrient-species samples designated "RC" were reported as "total" so that (a) the determination on a filtered sample reported as "dissolved" is distinct (without regard to sample preparation and analytical method), and (b) raw and filtered sample designations are consistent with those specified for other inorganic constituents. In addition, the U.S. Environmental Protection Agency (USEPA) required field collection of an unfiltered or "total" sample for these nutrients, and U.S. Geological Survey (USGS) projects using USEPA methods required an analogous procedure and laboratory schedule. To the best of our knowledge, however, USEPA protocols require filtration of particulate-laden samples in the laboratory prior to analytical determinations. In contrast, filtration of particulate-laden samples at the NWQL is discretionary. The chronology of methods used by USGS laboratories for determinations of total and dissolved nutrient species is partially documented as internal reports and memorandums. The 1979 edition of Techniques of Water-Resources Investigations (TWRI), book 5, chapter A1 (Skougstad and others) first published methods used at NWQL since 1973 to determine "total" nutrients by the four-channel method. Differences between these methods and those listed in the subsequent TWRI edition (1989) and editions of the NWQL Services Catalog are incompletely defined. In each time period, however, the only apparent difference in the method between the "total" and the "dissolved" nutrient species was that unfiltered samples were decanted before analysis. For example, the analytical methods for nitrite and for nitrite plus nitrate determinations on unfiltered samples specify that the determinations be performed on the supernatant portion of the sample "by decanting a suitable portion from a well-settled sample" (Skougstad and others, 1979). Moreover, Skougstad and others (1979) discussed the interference of suspended material on the method for total orthophosphate as follows: "...as phosphorus is easily adsorbed on sediment, the orthophosphate recovered from the supernatant solution above a suspended-sediment solution after some time has elapsed may be less than the orthophosphate that would have been determined in the filtrate from a sample filtered at the time of collection. The amount recovered may also depend on the type of sediment (clay, sand, etc.)." STATISTICAL EVALUATION OF DATA A statistical analysis was performed by C.J. Patton (Methods Research and Development Program, NWQL) and Jeff Pritt (Quality Management Group, NWQL) of all calendar year 1989 data to compare paired values of "total" and "dissolved" samples using a sign test. The number of paired values involved in the analysis for each species is listed in table 1: Table 1.--Number of paired values of the FC and RC nutrient samples statistically analyzed ______________________________________________________ Nutrient species Number of paired values: FC and RC samples ______________________________________________________ Ammonia 4,228 Nitrite 643 Nitrite plus nitrate 1,459 Orthophosphate 1,025 ______________________________________________________ The data were analyzed twice, using graphical inspection and two statistics software packages. Branch of Systems Analysis staff carefully reviewed the methods applications and data interpretation, and confirmed the conclusions presented here. A USGS Open-File Report is planned for publication during fiscal year 1993 to provide a detailed description of the problem, the data analyzed, the statistical methods used, and the conclusions reached. Visual inspection of the plotted differences in data values for paired analyses [unfiltered ("total") versus filtered ("dissolved")] indicated that the mean difference was about zero. A sign test on the filtered-unfiltered pairs indicated that the medians were not significantly different from each other at the 95-percent confidence level. The conclusion is that the analytical values reported for pairs of unfiltered and filtered concentrations for the identified nutrient species are statistically indistinguishable. As a result, in July 1991, NWQL ceased to apply the internal quality-assurance criterion requiring a rerun on nutrient samples for which the "dissolved" exceeded the "total" concentration for sample pairs analyzed using the four-channel method. Instead, the NWQL has been applying a two-sided test to RC and FC sample pairs that requires respective concentrations for the pair to be equivalent within analytical variance. INSTRUCTIONS TO DIVISION PERSONNEL 1. Effective immediately, project personnel are instructed to discontinue requests for the laboratory analytical codes shown on table 2. These laboratory codes will be discontinued on January 1, 1993. Project personnel must check the laboratory schedules they are using to determine whether the laboratory codes listed on table 2 are included. Table 2.--Laboratory codes to be deleted from the NWQL catalog ________________________________________________________________ Nutrient species and bottle designation Laboratory codes: Routine Low level ________________________________________________________________ Ammonia, Total.....................RC LC0123 LC0836 Nitrite, Total.....................RC LC0302 LC0840 Nitrite plus nitrate, Total........RC LC0304 LC0839 Orthophosphate, Total..............RC LC0297 LC0838 ________________________________________________________________ 2. After January 1, 1993, the only RC samples to be analyzed will be those that request LC1686 for total phosphorus and (or) LC1688 for total Kjeldahl nitrogen (ammonia plus organic nitrogen). RC samples will not be run on the four-channel analyzer. Therefore, no determination is available from NWQL for "total" concentrations on the following individual species: ammonia, nitrite, nitrite plus nitrate, and orthophosphate. 3. All samples submitted to the laboratory for determination of nutrient species on the four-channel analyzer (ammonia, nitrite, nitrite plus nitrate, and orthophosphate) must be filtered in the field and the sample bottle labeled with an "FC" designation. 4. Effective immediately, project personnel are asked not to request reruns on nutrient determinations using the four- channel method based on a criterion that the "total" (RC, or unfiltered) concentration must equal or exceed the "dissolved" (FC, or filtered) concentration reported for the sample pair. 5. Measurement of unfiltered or filtered concentrations of phosphorus and Kjedahl nitrogen can be obtained by requesting the appropriate laboratory codes listed in table 3, for which rigorous procedures for complete sample digestion are applied using the modified Jirka method that was implemented by NQWL in 1992 (see Office of Water Quality Technical Memorandum 92.10). Table 3.--Laboratory codes to be used for determination of phosphorus and nitrogen concentrations by complete digestion of the unfiltered and filtered sample _________________________________________________________________ Nutrient determination Sample Laboratory designation codes _________________________________________________________________ Phosphorus unfiltered,digested RC LC1686 filtered,digested FC LC1685 Nitrogen, Kjeldahl (ammonia plus organic nitrogen) unfiltered,digested RC LC1688 filtered,digested FC LC1687 _________________________________________________________________ One 125-mL "FC" bottle of filtered sample is sufficient sample volume for determination of: nitrogen and phosphorus species on the four-channel analyzer, filtered phosphorus (LC1685), and filtered Kjeldahl nitrogen (LC1687). A separate 125-mL "RC" bottle of unfiltered sample is required for total phosphorus (LC1686) and total Kjeldahl nitrogen (LC1688) determinations. Division personnel are advised that it is invalid to make the following interpretations with respect to comparison of unfiltered and filtered four-channel nutrient data: (1) to attribute environmental significance to differences or similarities between concentrations for the "total" and "dissolved" determinations; (2) to suggest concentrations measured in the unfiltered sample represent the total concentration present in the sample for the nutrient species analyzed; and (3) to implement a quality-assurance strategy based on the premise that the concentration measured in the unfiltered sample must exceed or equal the concentration measured in the filtered sample. REFERENCE CITED Skougstad, M.W., Fishman, M.J., Friedman, L.C., Erdmann, D.E., Duncan, S.S., 1979, Methods for Determination of Inorganic Substances in Water and Fluvial Sediments, U.S. Geological Survey Techniques of Water-Resources Investigations, book 5, chapter Al, 545 p. David A. Rickert Chief, Office of Water Quality This memorandum does not supersede any Office of Water Quality Technical Memorandum, but refers to Office of Water Quality Technical Memorandum 92.10. Key Words: Laboratory methods, nutrient species, nitrite, nitrate, ammonia, orthophosphate, four-channel method. Distribution: A, B, S, FO, PO