Environmental Toxicology and Chemistry, Vol. 31, No. 2, pp. 377–386, 2012 # 2011 SETAC Printed in the USA DOI: 10.1002/etc.734
DEVELOPMENT AND APPLICATION OF WHOLE-SEDIMENT TOXICITY TEST USING IMMOBILIZED FRESHWATER MICROALGAE PSEUDOKIRCHNERIELLA SUBCAPITATA LI-JUAN ZHANG, GUANG-GUO YING,* FENG CHEN, JIAN-LIANG ZHAO, LI WANG, and YI-XIANG FANG State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou, PR China (Submitted 29 May 2011; Returned for Revision 10 August 2011; Accepted 2 October 2011) Abstract— A method for a whole-sediment toxicity test using alginate immobilized microalgae Pseudokirchneriella subcapitata was
developed using spiked sediments and applied to contaminated field sediment samples. For method development, a growth inhibition test (72 h) with algal beads was conducted for the sediments spiked with Cu or diuron. The method was validated by determining dose– response relationships for Cu and diuron in both fine-grained and coarse-grained sediments. The results of a spiked sediment toxicity test suggested that sediment particle size distribution (clay content) had a significant effect on the growth of P. subcapitata. The developed method using immobilized microalgae P. subcapitata beads was applied successfully in the toxicity test and toxicity identification evaluation (TIE) for the four field sediment samples. After a series of extractions with 0.01 M CaCl2 solution, acetone, and dichloromethane, the extracted sediment, which was shown to be nontoxic to algae, was used as the control and diluent for the same sediment in the whole-sediment toxicity test. The results showed that all investigated field sediment samples were found to be toxic to the immobilized algae P. subcapitata, with their median effective concentration (EC50) values ranging from 41.4 to 79.0% after 72 h exposure. In the whole sediment TIE, growth of P. subcapitata was improved to varying degrees after adding zeolite, resin, or activated charcoal, suggesting different contributions to toxicity from ammonia, metals, and organic contaminants in the tested sediments. Environ. Toxicol. Chem. 2012;31:377–386. # 2011 SETAC Keywords—Sediment toxicity
Algae
Immobilized algal bead
Toxicity identification evaluation
Pseudokirchneriella subcapitata
toxicity of the sediments would be affected by their sediment physiochemical characteristics. Nelson et al. [8] found that silt loam soil reduced toxicity more effectively than sand when used as a diluent of the same sediment in the test by H. azteca [8]. Adams and Stauber [9] developed an acute whole-sediment toxicity test with a benthic marine microalga based on inhibition of esterase activity and used flow cytometry to distinguish algae from Cu tailing particles [9]. Mauffret et al. [10] assessed the toxicity of linear alkylbenzene sulfonate (LAS)-spiked sediments by using a benthic diatom, Cylindrotheca closterium, and found that the test method showed good sensitivity and reproducibility [10]. Immobilized microalgae have the potential to overcome the difficulties encountered with free algae in a whole-sediment toxicity test. Bozeman et al. [11] developed a method for an aquatic toxicity test using alginate-immobilized algae and proved that immobilized algae could be used in an aquatic toxicity assay [11]. Moreno-Garrido et al. [12] used the calcium-alginate immobilized marine diatom Phaeodactylum tricornutum in a toxicity test of LAS-spiked sediment [12]; however, the sediment grain size and the stability of the bead were not mentioned. More studies are needed to assess the sediment toxicity test using alginate immobilized algae by considering various factors such as standard freshwater algae, preparation and stability of algal beads, influences of sediment particle size, and shaking of the flasks during the bioassay. Freshwater planktonic microalgae Pseudokirchneriella subcapitata is a standard species in toxicity tests. The immobilized algal beads containing P. subcapitata could be used to test the sediment toxicity through direct contact with sediment. The object of the present study was to develop a wholesediment toxicity assay with immobilized freshwater microalgae Pseudokirchneriella subcapitata and to offer a rapid, sensitive, easy, and inexpensive method that could be used
INTRODUCTION
Standardized whole-effluent and receiving-water toxicity test methods using a series of aquatic organisms such as algae, daphnia, lemna, and fish [1] have been used, but only a few whole-sediment toxicity test methods are available. Wholesediment toxicity tests using the amphipod Hyalella azteca and the midge Chironomus tentans are standardized, but the test organisms have long life cycles that can make the tests costly and labor-intensive [2]. Therefore, developing faster, easier, and more ecologically relevant whole-sediment toxicity testing methods is necessary. Algae are common test organisms and sensitive to many toxicants and thus are widely used in water toxicity assays [3,4]. However, fewer applications are available that use microalgae in sediment toxicity tests than those that use amphipods and midges because of the poor growth rate of algae and the difficulty in counting algal cells [5]. Efforts have been made in recent years to use microalgae for whole-sediment toxicity tests, and the works were mainly focused on free benthic marine microalgae. A toxicity test was performed on sand spiked with heavy metals using the marine benthic diatom Cylindrotheca closterium [6]. The method is suitable only for sandy sediment, which may be very different from natural sediment. Arau´jo et al. [7] applied this method to marine sediment quality assessment. During a sediment toxicity test, algal cells were counted in a Neubauer chamber with fluorescence microscopy. In this experiment, acid-washed ground sand was used as the control for four different sediment samples. Using the same control for completely different samples is not appropriate because the * To whom correspondence may be addressed (
[email protected]). Published online 7 November 2011 in Wiley Online Library (wileyonlinelibrary.com). 377
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to evaluate the toxicity of freshwater sediment. The algal immobilization procedure was optimized to produce beads with good algal growth and stability. The prepared algal beads were tested for both fine and coarse sediments spiked with a metal ion Cu and a herbicide, diuron. The effect of clay content in sediment on the growth of immobilized algae and shaking of flasks during the bioassay were also assessed. The developed method was then applied in a toxicity assay of four field sediment samples, and toxicity identification evaluation (TIE) was performed to characterize the toxicants in the sediments. MATERIAL AND METHODS
Chemicals and reagents
Alginic acid sodium salt from brown algae (No.71238) and activated charcoal were supplied by Sigma. A weak acidic cation resin Lewatit TP 207 was obtained from Fluka, and zeolite was purchased from National Pharmaceutical Group Chemical Reagent. The stock solution of Cu in the form of CuSO4 was prepared in the Milli-Q water at a concentration of 20,000 mg/L, whereas the stock solution of diuron (from the Shanghai Pesticide Research Institute, 98% mg/L) was prepared at 10,000 mg/L in methanol. All inorganic chemicals used in the test were of analytical grade and purchased from the Guangzhou Chemical Factory, China; all organic solvents were of highperformance liquid chromatography grade and obtained from Merck. Sediment collection, preparation, and characterization
Two sediments (S1 and S2) with different grain sizes were prepared for the spiking experiment. Sediment S1 was a finegrained, natural freshwater sediment (predominantly 66% silt and 29% clay), collected from the Liuxi reservoir located to the northeast of Guangzhou city in southern China. Natural sand used to prepare artificial sediment S2 was obtained from Dongguan canal, sieved through an 80- to 200-mesh sieve, then washed with 10% HNO3 and organic solvents (methanol and acetone) to remove extractable metals and organic contaminants, and finally, rinsed with Milli-Q water several times. Physiochemical characteristics of sediment S1 and washed sand are shown in Table 1. Sediment S2 was an artificial coarsegrained sediment with 2.9% clay (
