Spectrophotometric methods for the determination of photosynthetic pigments in stratified lakes: a critical analysis based on comparisons with HPLC determinations in a model lake

Spectrophotometric methods for the determination of photosynthetic pigments in stratified lakes: a critical analysis based on comparisons with HPLC determinations in a model lake High-performance liquid chromatography (HPLC) is an accurate method for photosynthetic pigment analysis; however, spectrophotometric equations are also frequently used for pigment quantification in aquatic systems. Here, we present a critical analysis of the most-used spectrophotometric equations by comparing the results obtained using these equations with unambiguous HPLC determinations. The study was performed in Lake La Cruz (central Spain). In this meromictic lake with strong thermal stratification, photosynthetic populations occur in different, vertically stratified layers. Eukaryotic algae and picocyanobacteria are mostly located in oxic layers, whereas purple sulphur bacteria grow at the oxic-anoxic interface and below, and green sulphur bacteria occur primarily in deeper anoxic layers. This broad diversity of photosynthetic microorganisms involves a complex mixture of photosynthetic pigments that often exhibit overlapping absorption spectra. We tested spectrophotometric equations using samples that represented the entire range of spatial and temporal variability of the lake. For chlorophyll-a, the best correlations with all tested equations were observed for oxic layers. Regardless of where the sample was obtained, the best fit for chlorophyll-a was produced by the equation of Overmann & Tilzer, which is specifically designed to handle mixtures of chlorophyll-a and bacteriochlorophyll-d from green sulphur bacteria. Trichromatic equations for determining chlorophyll-b and -c exhibited strong interferences in anoxic waters, whereas in the upper layers of the lake, concentrations of these pigments were usually below the detection limit, restricting the use of these equations. The equations of Takahashi & Ichimura for bacterial pigments slightly overestimated both bacteriochlorophyll-a and -d by approximately 10 % and underestimated bacteriochlorophyll-c by nearly 23 %, although for bacteriochlorophyll-d, the correlation was better than those obtained using the dichromatic equations of Parkin & Brock and Overmann & Tilzer, respectively. Total carotenoid abundance can be assessed with the equation designed for this purpose by Strickland & Parsons (1972); however, the accuracy of the results differs with depth and is strongly biased by the presence of the bacterial carotenoid okenone. On the other hand, dual-wavelength carotenoid-to-chlorophyll-a ratios (430/665 and 480/665) only produced acceptable results in the epilimnion, with the occurrence of the bacterial carotenoid okenone in the microaerobic and anoxic layers again producing significant interference. Additionally, the wide variation with depth of the carotenoid composition undermined the validity of these dual-wavelength ratios. In conclusion, our findings indicate that a complete and unambiguous study of photosynthetic pigments in highly stratified lakes with overlapping populations of phototrophic microorganisms requires the use of HPLC techniques. Anyway, our results demonstrate that chlorophyll-a and bacteriochlorophyll-a and -d can be directly measured in oxic and anoxic layers, respectively, using spectrophotometric methods with an error lower than 10 %. However, according to our results, chlorophyll-b and -c and bacteriochlorophyll-c cannot be accurately estimated by spectrophotometric methods in stratified lakes.