Attachment

INFORMATION BRIEFING

ACTION For information. A recent quality assurance review found analytical errors in the chlorophyll measurement method used by the MWRA outfall monitoring program during 1998-2000. This briefing describes the steps we are taking to correct the errors and to prevent their recurrence. This briefing describes technical issues related to chlorophyll data obtained using a lab fluorometer to analyze sample filters.

Technical issues and corrections. In our evaluation of the fluorescence and bottle chlorophyll data from Fall 2000, the project team identified two technical issues requiring action. Both issues relate to the standard solution that was used to calibrate the fluorometer. Each issue leads to an upward bias in the extracted chlorophyll data.

In the laboratory, chlorophyll a is measured by fluorometric analysis of acetone-extracted filters before and after acidification (Albro et al. 1998). The method used in MWRA’s program from 1998-2000 refers to an SOP (Standard Operating Procedure; Battelle 1999) and to EPA method 445.0 (Arar and Collins 1992) for detail. The SOP checks for degradation of the chlorophyll standard solution during the acidification step and determines the concentration of the standard solution from the weight of chlorophyll reagent powder added to a known volume of solvent; this assumes that the purchased reagent is of known weight purity as supplied by the manufacturer. A spectrophotometer should have been used to determine the purity and concentration of the chlorophyll standard, but this was not done.

The analytical problems resulted from:

1. Degraded standard. By mistake, a chlorophyll standard that was degraded was used to calibrate the samples from late September 2000 and October 2000 water column surveys (the period of the fall bloom). The error led to an upward bias of about 40%. We have corrected the error using a recommendation (Jay O’Reilly and Kristen Kohlberg pers. comm.) to numerically recalibrate the dataset on the phaeophytin of the acidified standard.
   
   
2. Impurity of standard. The project team mistakenly believed that the standards we were using were pure chlorophyll a. We now know that the standards were not pure, and that the manufacturer’s stated purity varied by lot number (90.7% or 94.8% depending on lot number). Furthermore, the purchased chlorophyll standard appears to have variable and unknown weight purity from ampule to ampule when we evaluated this with a spectrophotometer. We speculate that this is due to differences in dryness of the reagent due to imperfectly sealed ampules. We will evaluate this further on additional ampules, and examine our data on a secondary standard (coproporphyrin) that was run with every batch of analyses. The purity issues may affect all data from 1998 to the end of 2000.

The chlorophyll data from 1992-1997 were measured using a spectrophotometer-checked standard and should be immune to the purity issue. We are nevertheless expanding our review to include those years. We are extending the review to ensure that the entire chlorophyll baseline is fully comparable and as error-free as feasible.

We are immediately reviewing and revising our SOP for chlorophyll analysis to ensure that the purity of the standard is known and certified.

These investigations are ongoing, especially the evaluation of the variability in purity of the purchased chlorophyll standard. In situ fluorescence data, from which the chlorophyll thresholds are calculated (see accompanying briefing on thresholds calculation), are calibrated against the results of these extracted chlorophyll samples and are therefore also under review. Until we have the results of that evaluation, MWRA cannot validate the calibrated fluorescence data for the purposes of calculating either the chlorophyll baseline (data through August 2000) or for testing the Fall 2000 seasonal chlorophyll threshold. We anticipate completing this evaluation and reporting to OMSAP within 60 days.

REFERENCES

Albro CS, Trulli HK, Boyle JD, Sauchuk SA, Oviatt CA, Keller AA, Zimmerman C, Turner JT, Borkman D, Tucker J. 1998. Combined work/quality assurance plan for baseline water quality monitoring: 1998-2000. Boston: Massachusetts Water Resources Authority. Report ENQUAD ms-048. 121 p. http://www.mwra.state.ma.us/harbor/enquad/pdf/ms-048.pdf

Arar EJ, Collins GB. 1992. In Vitro Determination of Chlorophyll a and Phaeophytin a in Marine and Freshwater Phytoplankton by Fluorescence. Method 445.0 Version 1.1 (November 1992). U.S. Environmental Protection Agency, Environmental Monitoring Systems Laboratory, Office of Research and Development, Cincinnati, OH. http://www.epa.gov/nerlcwww/marinmet.htm

Battelle. 1999. SOP No. 5-265, Extraction and Analysis of Chlorophyll a and Phaeophytin a in Seawater using a Turner [Designs] Model 10AU Fluorometer.

Onboard Sample Processing for Chlorophyll a and Phaeophytin

Laboratory Sample Processing and Analysis for Chlorophyll a and Phaeophytin

Calibration procedures and preventive maintenance