Article pubs.acs.org/est
Determination and Occurrence of Retinoids in a Eutrophic Lake (Taihu Lake, China): Cyanobacteria Blooms Produce Teratogenic Retinal Xiaoqin Wu, Jieqiong Jiang, and Jianying Hu* MOE Laboratory for Earth Surface Process, College of Urban and Environmental Sciences, Peking University, Beijing 100871, China S Supporting Information *
ABSTRACT: Besides retinoic acids (RAs), some retinoids such as retinal (RAL) and retinol (ROH), which are considered as RA precursors in vertebrates, are also reported to be teratogenic agents. In this study we investigated four RA precursors including RAL, ROH, retinyl palmitate, and β-carotene in the eutrophic Taihu Lake, China, by developing a sensitive analytical method. RAL and β-carotene were widely detected in natural cyanobacteria blooms and lake water. Intracellular concentrations of RAL and β-carotene in blooms were 9.4 to 6.9 × 103 and 3.4 to 1.8 × 105 ng L−1, respectively, and their concentrations in lake water were up to 1.4 × 10 ng L−1 (RAL) and 9.8 × 102 ng L−1 (βcarotene). The good correlation between intracellular concentrations of RAL and RAs implied that RAL was involved in the production of RAs by cyanobacteria blooms. Further examination of 39 cyanobacteria and algae species revealed that most species could produce RAL and β-carotene. The greatest amount of RAL was found in Chlamydomonas sp. (FACHB-715; 1.9 × 103 ng g−1 dry weight). As the main cyanobacteria in Taihu Lake, many Microcystis species could produce high amounts of RAL and were thought to greatly contribute to the production of RAL measured in the blooms. Productions of RAL and β-carotene by cyanobacteria were associated with species, origin location, and growth stage. The results in this study present the existence of a potential risk to aquatic animals living in a eutrophic environment from a high concentration of RAL in cyanobacteria blooms and also provide a clue for further investigating the mechanism underlying the biosynthetic pathway of RAs in cyanobacteria and algae.
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Lake, China.12 Although the biosynthetic pathway of RAs in cyanobacteria and algae remains unclear, considering the widespread presence of carotenoids in cyanobacteria and algae, and the reports of RAL acting as photoreceptors in some green algae and cyanobacteria,13−16 it can be inferred that these RA precursors should contribute to the production of RAs in cyanobacteria and algae and should also exist in blooms in eutrophic environment. So far, only RAs have been studied for their occurrences and sources in the environment,5,7,12 leaving the other retinoids, especially RA precursors, uninvestigated. A major hurdle for studying the RA precursors in the environment is the lack of a sensitive analytical method. While liquid chromatography coupled to mass spectrometry (LC-MS or LC-MS/MS) or an ultraviolet detector (LC-UV) is generally used for the determination of retinoids and carotenoids in biological samples,17−26 an effective sample extraction procedure needs to be developed for the analysis of environmental water samples because of the low levels of retinoids in the environment. To demonstrate the hypothesis that RA precursors exist in the blooms of a eutrophic environment, four RA precursors including RAL, ROH, RPT, and β-carotene were determined
INTRODUCTION Retinoids are a class of compounds including vitamin A (retinol, ROH) and its natural analogues such as retinal (RAL) and retinoic acids (RAs), in addition to its synthetic derivatives.1 During the past decade, environmental retinoids, especially RAs, have become an increasing concern due to their teratogenecities and a potential role in causing deformed amphibians in the field.2−7 As one of the most potent known animal teratogens, RAs are generally thought of as vertebrate-specific hormones and can be transformed in vivo from RAL, ROH, retinyl esters (REs), and carotenoids,1 which are collectively called RA precursors. The RA precursors are largely synthesized and widely used in medicines, nutrition supplements, feed additives, the food industry, and cosmetics. It has been extensively proved that an excess of some RA precursors can produce adverse effects in humans and animals.8 For example, oral administration of retinyl palmitate (RPT) to cynomolgus monkey during early pregnancy can cause abortion and malformation in the embryo.9 Also, ROH can cause limb malformation in tadpoles with tail amputation,10 and exposure to RAL and ROH can generate various kinds of deformities in Zebrafish.11 Besides endogenous origin in vertebrates as well as artificial synthesis, RAs (including their analogues 4-oxo-RAs) were recently found in some laboratory-cultured cyanobacteria and algae species which are commonly observed in the aquatic environment, as well as in natural cyanobacteria blooms in Taihu © 2012 American Chemical Society
Received: Revised: Accepted: Published: 807
September 3, 2012 December 16, 2012 December 20, 2012 December 20, 2012 dx.doi.org/10.1021/es303582u | Environ. Sci. Technol. 2013, 47, 807−814
Environmental Science & Technology
Article
in Taihu Lake, the eutrophic lake we investigated previously,12 by using a unique analytical method developed in this paper. Further examination of 39 representative cyanobacteria and algae species was carried out to determine the specific species that mainly contributed to the production of these RA precursors in cyanobacteria blooms.
algae including 24 cyanophyta, 8 chlorophyta, 6 bacillariophyta, and 1 euglenophyta with the cell numbers more than 108 cell L−1 were purchased from the Freshwater Algae Culture Collection of the Institute of Hydrobiology (FACHB), Chinese Academy of Sciences. To compare the species-specific production of RAL and β-carotene, the cultures of cyanobacteria and algae at a similar growth stage, i.e., logarithmic growth stage, were used for analysis. Two axenic cultures of cyanobacteria, Microcystis aeruginosa (PCC 7806) and Synechococcus sp. (PCC 7002), were from the Pasteur Culture Collection of Cyanobacteria (PCC), France. Laboratory cultivation of Microcystis f los-aquae (FACHB-1028) was as described in our previous paper.12 Briefly, the cyanobacteria strain consisting of three replicates was grown in BG11 medium at 25 °C on a 14 h light/10 h dark cycle. At each sampling time, the cell counting was carried out (n = 6) for each sample and the number of cells (cell L−1) was calculated. The culture (35 mL) was centrifuged at 6000 rpm for 10 min, and then the cyanobacteria/algae pellets were resuspended in 2− 3 mL of phosphate buffer (0.2 M, pH = 6.0) before performing cell disruption as described above. After adding RAL-d5 as surrogate standard, the sample was extracted three times with 5 mL of hexane/chloroform (3:1, v/v) by mechanically shaking for 5 min, and the combined organic layer was evaporated to dryness under a gentle stream of nitrogen gas. The residue was dissolved in 350 μL of acetonitrile and then injected into the LC-MS/MS system. Instrumentation. Chromatographic separation of ROH, RAL, and RPT was carried out on an ACQUITY ultraperformance liquid chromatography (UPLC) BEH Phenyl column (2.1 × 100 mm, 1.7 μm, Waters) at a flow rate of 0.3 mL min−1 at 40 °C, using a Waters ACQUITY UPLC system consisting of binary solvent manager and sample manager. Mobile phase A was methanol, and mobile phase B was 0.1% formic acid in ultrapure water. The gradient (with respect to mobile phase A) was as follows: 0−1 min, 20−70% A; 1−4 min, 70−100% A; 4−6 min, 100% A; 6−8 min, re-equilibration with 20% A. The injection volume was set to 5 μL. Chromatographic analysis of β-carotene was carried out on an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm, Waters) at a flow rate of 0.3 mL min−1 and 40 °C, using the same Waters UPLC system. Mobile phase A was methanol, and mobile phase B was 0.1% formic acid in ultrapure water. The gradient (with respect to mobile phase A) was as follows: 0−1 min, 20− 70% A; 1−6 min, 70−100% A; 6−14 min, 100% A; 14−16 min, re-equilibration with 20% A. The injection volume was set to 5 μL. The analysis was performed using a Waters Micromass Quattro Premier XE detector equipped with an electrospray ionization (ESI) source. All analytes were determined in positive ion mode (ESI+) by multiple reaction monitoring (MRM), and the optimized parameters are listed in Table S1, Supporting Information. The optimized parameters were as follows: capillary voltage, 3.40 kV; cone voltage, 25.00 V; extractor, 5.00 V; RF lens, 0.5 V; source temperature, 110 °C; desolvation temperature, 350 °C; cone gas flow, 50 L h−1; desolvation gas flow, 600 L h−1; multiplier voltage, 650 V. Argon (99.999%) was used as the collision gas, and argon pressure in the collision cell was kept at 3.5 × 10−3 mbar. All data were acquired and processed using MassLynx 4.1 software. Quality Assurance and Quality Control (QA/AC). Confirmation of the target analytes in the environmental samples was accomplished by comparing the retention time (
