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Keywords:

  • Sediment toxicity;
  • Algae;
  • Immobilized algal bead;
  • Toxicity identification evaluation;
  • Pseudokirchneriella subcapitata

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. REFERENCES

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


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. REFERENCES

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. Whole-sediment 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 closterium6. The method is suitable only for sandy sediment, which may be very different from natural sediment. Araújo 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 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. azteca8. 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 whole-sediment toxicity assay with immobilized freshwater microalgae Pseudokirchneriella subcapitata and to offer a rapid, sensitive, easy, and inexpensive method that could be used 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

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. REFERENCES

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 high-performance 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 fine-grained, 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 coarse-grained sediment with 2.9% clay (<0.002 mm), which was prepared by mixing 10% of sediment S1 with 90% of the acid washed sand by weight.

Table 1. Physiochemical characteristics of the sediment samples
ParametersSediment samples
S1Washed sandABCD
  1. ND = below the detection limit; TOC = total organic carbon; PAHs = 16 U.S. Environmental Protection Agency (U.S. EPA) polycyclic aromatic hydrocarbons; OCPs = U.S. EPA organochlorine pesticides.

pH6.056.356.576.306.856.65
TOC (%)1.4003.492.252.521.98
Clay (<0.002 mm, %)2903598
Silt (0.002–0.063 mm, %)66023275857
Sand (>0.063 mm, %)510074683335
Moisture content (%)590.577626457
NHmath image-N (µg/g dry wt)9ND73677564
Metals (µg/g dry wt)
 Cu110.72492,4201,83734
 Zn795.73922,0091,370106
 Pb500.2714850321954
 Cr201.58622,7761,48152
 Mn426.23563433622601
 Ni201.39301,13548729
Organic compounds (ng/g dry wt)
 ΣPAHs3,766ND2,3042,2441,2545,756
 ΣOCPsNDND19500.830.77
Current use pesticide
  IsoprocarbNDND1.01NDNDND
  CarbofuranNDND13,9867,13014,1985,143
  PhoximNDND2.40.410.400.34
  CyanazineNDND3,7471,5832,6921,326
  CyfluthrinNDND3294392,7782,748
  LinuronNDNDNDND875754
  PyridabenNDNDNDND2519
  DimethoateNDNDNDND74107
  Deisopropylatrazine28NDNDNDNDND
  Iprobenfos69NDNDNDNDND
  Chlorfenapyr7.6NDNDNDNDND
  Fenaminosulf32NDNDNDNDND
Pharmaceuticals and personal care products
  Bisphenol ANDND230529658.6
  TriclocarbanNDND2631365820
  Nonylphenol activatedNDND1031053519
  Salicylic acidNDND9.5244624
  CarbamazepineNDND0.250.240.490.29

To assess the effect of clay content on the growth of algae, a series of artificial sediments with different percentages of clay (<0.002 mm) were also prepared by mixing the acid-washed sand with sediment S1 in various proportions. Artificial prepared sediments were verified to be free of bioavailable contaminants.

Four surface sediment samples were collected in April 2009 from four heavily polluted rivers in the Pearl River Delta Region for whole-sediment toxicity tests. The four field sediments were named as sediment A (Hanxi River, 23°02′53″N, 113°52′32″E), sediment B (Danshui River, 22°46′47″N, 114°25′08″E), sediment C (Longgang River, 22°46′03″N, 114°20′07″E), and sediment D (Shima River, 22°44′16″N, 114°03′34″E). Once collected, the four fresh sediments were transported to the laboratory and stored in the dark at 4°C. The sediment toxicity test was conducted within four weeks. The water contents of the fresh sediments were determined by drying the wet sediment at 105°C for 12 h. Ammonia in the pore water of the fresh sediments was measured by Nessler's reagent colorimetric method 13.

Aliquots of the collected sediments were freeze-dried and characterized for chemical and physical properties and contaminant levels. Sediment pH was measured in 0.01 M CaCl2 solution (sediment to solution ratio of 1:5, w/v) using a pH meter. Total organic carbon (%) was measured with an LECO carbon analyzer after removal of carbonates with HCl, and sediment particle size distribution was analyzed by using the pipette method. Inorganic and organic contaminants in the sediments were also determined using the analytical methods available in our laboratory (Table 1). Concentrations of heavy metals (Pb, Cr, Cu, Zn, Ni, and Mn) in the sediments were analyzed by inductively coupled plasma mass spectrometry. Polycyclic aromatic hydrocarbons in the sediments were determined using the method of Barco-Bonilla et al. 14, and some currently used pesticides and organochlorine pesticides were measured using the method of Yang et al. 15. Other emerging contaminants were analyzed using the method of Chen et al. 16.

In the spiked sediment toxicity test, Cu concentrations in the overlying water and sediment of a test sample were determined using an atomic absorption spectrophotometer (Hitachi Z-2300) using the method of Suedel et al. 17, and the concentrations of diuron were determined with the method of Gatidoua et al. 18.

Algae cultures

The freshwater green alga Pseudokirchneriella subcapitata Hindak (formerly known as Selenastrum capricornutum Printz) was chosen because it is a standard species in toxicity tests. The test algal species P. subcapitata was obtained from the Algae Culture Collection of Wuhan Institute of Hydrobiology, Chinese Academy of Sciences. The U.S. Environmental Protection Agency (U.S. EPA) medium 19 was prepared with ethylenediaminetetraacetic acid (EDTA) for culturing and without EDTA for algal bioassays to avoid the effect of the EDTA on heavy metals. Pseudokirchneriella subcapitata cultures were maintained in clean, sterilized 250-ml glass Erlenmeyer flasks topped with synthetic cotton and incubated in 100 ml U.S. EPA stock medium with EDTA (sterilized by sterile filtration, 0.22 µm) at 24 ± 1°C, under continuous illumination (4,000 lux, cool white fluorescence) 19. The cultures were renewed weekly using an aseptic technique to avoid bacterial contamination.

Algae immobilization

The algal calcium-alginate beads were prepared before the test setup. A 3- to 4-d-old P. subcapitata culture in log phase growth was used for immobilization. The immobilization procedure was adopted from Bozeman 11 with some changes in composition of alginate and CaCl2 to obtain algal beads with good algal growth and stable structure. Concentrations of sodium alginate (3, 4, and 5%) and CaCl2 · 2H2O solution (2, 3, and 4%) were optimized in the preliminary experiment. Beads were cultured with the U.S. EPA medium without EDTA. The growth of algae in beads was measured, and the stability of the bead was tested using the culture medium supplemented with 100 mg/L NaH2PO4 · 2H2O, because the sodium and phosphate can cause bead disruption or dissolution 20. Three replicates, with 10 beads each, were used in each treatment. The experiment was carried out in 150-ml glass vials containing 50 ml medium and incubated for 72 h at 24 ± 1°C under continuous illumination (4,000 lux, cool white fluorescence). The diameter of each bead was measured after 72 h incubation under a microscope, with the aim to determine bead stability (swelling or shrinkage).

The optimal concentration of 4% sodium alginate and 4% CaCl2 · 2H2O solution was chosen and used for further whole-sediment toxicity testing experiments. Each bead had approximately 0.037 ml containing 5 × 104 cells. The beads were stored in the dark at 4°C until use. The outer and internal morphology of blank Ca-alginate beads without algae and beads with P. subcapitata after incubation for 5 d in the U.S. EPA medium were directly observed under a scanning electron microscope (FEI Quanta 400).

Effect of clay content and shaking

Artificial sediments with different percentages of clay (1.5, 3, 6, 12, 18, 24, and 29%) were tested. During 72 h incubation, the effects of shaking and not shaking the flasks were also considered. For the shaking treatment, the flasks were shaken by hand twice a day and re-randomized. For the without-shaking treatment, the flasks were incubated statically and re-randomized twice daily carefully to avoid any turbulence. A growth inhibition test with algae beads for these treatments was performed by following the 72-h exposure experiment protocol. Three replicates were used for each treatment and medium control without sediment (CK1) and 100% sand control (CK2).

Seventy-two-hour exposure experiment protocol

For 72-h exposure experiment, 1 g (dry wt) prepared sediment was placed in each flask (150 ml), followed by an addition of 50 ml U.S. EPA medium (without EDTA), and then the flask was thoroughly mixed. The flasks topped with gauzes were placed in an incubator at the test temperature (24°C) for 24 h to equilibrate. After 24 h, 10 beads containing 5 × 104 cells/bead were added to each flask, being careful to avoid turbulence at an initial concentration of approximately 1 × 104 cells/ml. Then the flasks were placed randomly in an incubator and incubated at 24 ± 1°C under continuous illumination (4,000 lux, cool white fluorescence) for 72 h. After 72-h exposure, the 10 beads of 0.037 ml/bead in each flask were taken out and dissolved overnight in 1.63 ml sodium citrate (3%, w/v), which has a high affinity for calcium at 4°C. The algae cell concentration (cell yields used as the endpoint) was determined by counting algal cells on a hemocytometer under a microscope. The variability of the algal counting (n = 3) was less than 15%.

Blank control (sediment control) and solvent controls (trace methanol, <0.1%) were always tested alongside the test sediments. The test was performed in six replicates for each concentration treatment; three replicates were used to determine cell yield after 72 h exposure, and the other three replicates were used to determine pH, dissolved oxygen, and Cu or diuron in overlying water and sediment on day 0 and day 3.

Toxicity of a sediment to algae in beads was measured on the basis of cell yield. Percentage inhibition of algal growth was calculated using the following equation

  • equation image

where I is the percentage inhibition of algal growth for each test concentration replicate; Rc is the mean cell yield for the control, and R is the cell yield for each test concentration replicate.

Spiked sediment toxicity test with P. subcapitata

Two sediments (S1 with 29% clay, and S2 with 2.9% clay) spiked individually with the two model compounds (Cu and diuron) were selected for whole-sediment toxicity test trials. To determine sigmoidal dose–response relationships, a series of concentrations for each compound were spiked in the sediments. Copper in the form of CuSO4 at the nominal concentrations of 0, 94, 188, 375, 750, 1,500, 3,000, 6,000, and 12,000 mg/kg (dry wt) was spiked into sediment S1, whereas Cu at the nominal concentrations of 0, 24, 48, 94, 188, 375, 750, and 1,500 mg/kg dry weight was spiked into sediment S2. The diuron-spiked sediments were prepared at eight nominal concentrations of 0, 0.4, 0.8, 1.6, 3.2, 6.3, 12.5, 25, and 50 mg/kg (dry wt) for sediment S1, and at the nominal concentrations of 0, 0.25, 0.5, 1, 2.5, 5, 10, and 20 mg/kg (dry wt) for sediment S2 according to the spiking procedure from the U.S. EPA and Simpson et al. 21, 22. The required volume of the stock solution (copper or diuron) was added directly to the 400 g fresh sediment at a sediment to water ratio of 4:1. Then the sediment was mixed thoroughly for 2 h, equilibrated for 24 h, and neutralized to pH 7 with NaOH. The spiked sediments were finally held in the dark at 4°C for a month to equilibrate, and their pH values were checked every day and adjusted if necessary to pH 7.

The whole sediment toxicity test was performed by following the 72-h exposure experiment protocol as described. The effects of shaking the flasks twice per day or not shaking the flasks were also considered during the 72-h test. Blank control (medium control and unspiked sediment control) and solvent controls (trace methanol, <0.1%) were also tested alongside the spiked sediments. The test was carried out in six replicates for each concentration treatment, three replicates used to determine cell yield after 72 h exposure and the other three replicates to determine pH, dissolved oxygen, and Cu or diuron in overlying water and sediment on day 0 and day 3. The 72-h bioassay was performed twice in succession. The water-only test was also included for quality assurance as described in the following section.

Water-only test with immobilized P. subcapitata bead

Pseudokirchneriella subcapitata beads were exposed to Cu (0, 5, 10, 20, 40, 80, and 160 µg/L) and diuron (0, 5, 10, 20, 40, 80, and 160 µg/L), respectively, dissolved in water, then in the sediment tests as described. At least three concentrations of Cu or diuron were included in each P. subcapitata bioassay to ensure that each batch of algae was responding to a known toxicant in a reproducible manner.

Field sediment toxicity test and TIE

To perform whole-sediment toxicity tests for the field-collected sediments, corresponding control sediment and diluent sediment were prepared before the testing experiment. The sediment, from which organic and metal contaminants were almost extracted, could be regarded as a clean sediment and was used as the control or diluent sediment in the whole-sediment toxicity test. The diluent of each field sediment sample was prepared as follows.

An aliquot of 5 g (dry wt) sediment sample was extracted three times with 25 ml 0.01 M CaCl2 solution by shaking for 4 h each time and then centrifuged. The supernatant of CaCl2 solution was removed. The sediment was washed with Milli-Q water three times and centrifuged to remove CaCl2. After this, the remaining sediment was again extracted three times with 15 ml acetone and 15 ml dichloromethane by ultrasonication at room temperature (15 min each time). The extracted sediment was removed to a dish and put in the fume hood for 24 h to allow the solvents to volatilize. Finally, the sediment was mixed with Milli-Q water and shaken for 24 h to substantially rehydrate the sediment and make its water content the same as that in the untreated field sediment.

To determine whether the four diluent sediments were toxic to P. subcapitata, the toxicity of the diluent sediment elutriates was evaluated with free algae P. subcapitata in a 96-well microplate 23. Sediment elutriate was prepared as follows according to Haring et al. 24.

Whole-sediment tests with immobilized P. subcapitata beads were conducted to determine the toxicity of the four contaminated sediment samples. Each sediment sample was diluted with its corresponding control sediment prepared as described at a percentage of 100, 75, 50, and 25% on a final dry weight of 1 g sediment. Then the prepared sediments were washed into 150-ml flasks with 50 ml medium each. After being thoroughly mixed, the flasks were incubated without shaking during the 72-h assay. Three replicates were used for each level of the prepared sediments as well as for the control sediment. The initial sediment toxicity test was performed according to the developed protocol as described previously.

Further toxicity tests were performed to determine the toxic classes (ammonia, metals, and organics) in the sediments. The whole sediment TIE phase I included addition of 10% zeolite, 10% cation exchange resin, and 5% activated charcoal, which were targeted at three types of toxicants commonly found in sediments: ammonia, cationic metals, and organic chemicals, respectively 25. Each manipulation was performed in triplicate, and the mixture was shaken well and held at 4°C in the dark for 24 h. The growth inhibition test with algal beads was conducted to determine whether toxicity changes occurred in each manipulation. A simultaneous test of unmanipulated sediment (baseline toxicity test) was performed, and the baseline toxicity test was conducted in triplicate alongside for comparison.

Statistical analysis

For the spiked sediment toxicity test, EC50 values (calculated concentration at which there was a 50% reduction in cell yield compared with controls) of the algal growth inhibition test were calculated using sigma plot, and dose–response curves were fitted to a four-parameter logistic equation with the Sigmaplot software (Ver. 10.0), whereas EC50 values (percentage of field sediment that inhibits growth yield by 50%) of the field sediment toxicity test were calculated using an EC50 calculator program developed by the Commonwealth Scientific and Industrial Research Organisation (CSIRO, Adelaide, Australia) 26. SPSS Version 14 software was employed for statistical analysis. One-way analysis of variance was used to compare any significant differences (p < 0.05) in growth inhibition between individual treatments and the control, enabling a calculation of no-observable-effect concentration and lowest-observable-effect concentration. Averages and standard deviations were calculated with Microsoft Excel 2003. Measured concentrations of Cu and diuron were used in calculation of EC50, no-observable-effect concentration, and lowest-observable-effect concentration.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. REFERENCES

Algal bead optimization

Algal beads were optimized based on cell growth and bead stability by varying concentrations of sodium alginate and CaCl2 · 2H2O solution. As shown in Table 2, the beads prepared with 4% alginate solution and hardened with 4% CaCl2 · 2H2O solution were found to be quite stable, with only 2.1% change in diameter after 72 h exposure to NaH2PO4 (100 mg/ L). Each bead obtained under this condition was 3,905 ± 25 µm in diameter and contained 52,000 ± 2,400 cells, which allowed approximately 18-fold growth of P. subcapitata cells after 72-h exposure to the U.S. EPA medium (without EDTA) used in the present study. A scanning electron micrograph image (Fig. 1) of the blank bead and calcium-alginate beads containing P. subcapitata after 5-d incubation showed that the algae strain distributed well in the bead material (calcium alginate), and the cell density of P. subcapitata in each bead increased whether in the internal and external parts of the bead after incubation in the growth medium.

Table 2. Growth of Pseudokirchneriella subcapitata cells in bead and stability of beads prepared using different alginate solutions and CaCl2 solutions
No.Alginate (%w/v)CaCl2 · 2H2O (%w/v)Cell density (104 cells/ml) (mean ± standard deviation)Bead diameter change after 72 h (%)
13222.5 ± 1.28.0
23321.9 ± 0.87.0
33420.5 ± 0.35.4
44222.5 ± 0.76.2
54318.9 ± 0.33.7
64418.5 ± 0.32.1
75217.3 ± 0.34.1
85316.9 ± 0.62.4
9548.9 ± 0.30.8
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Figure 1. Scanning electron micrograph image blank Ca-alginate beads without algae and beads with Pseudokirchneriella subcapitata after incubation for 5 d in U.S. Environmental Protection Agency medium. (A) Whole blank Ca-alginate bead; (B) whole bead with P. subcapitata; (C) blank bead: intact outer surface of the bead; (D) bead with P. subcapitata: intact outer surface of the bead; (E) blank bead: internal of the bead; (F) bead with P. subcapitata: internal of the bead. Magnification is indicated individually on data bar at the bottom of micrograph.

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Effect of clay content and shaking

Preliminary tests showed that the elutriates of sediments S1 and S2 were not toxic to free algae P. subcapitata. The percentage inhibition of the elutriates of S1 and S2 to free algae P. subcapitata were 0.3 ± 4.6 and 0.7 ± 3.7%, respectively. No significant differences were found between them and medium control (0 ± 4.9%) (p > 0.05). At the same time, no significant difference in toxicity was found between extracted and unextracted sediment S1, suggesting the contaminants in the sediment were not bioavailable. The growth of immobilized P. subcapitata was influenced by sediment S1 compared with that without sediment addition. Figure 2 shows the populations of P. subcapitata after 72-h exposure to 1 g (dry wt) of the mixed sediments with different clay contents in two shaking methods during the assay. The average population of algal cells in each treatment ranged from 3.6 × 104 to 18.4 × 104 cells/ml after 72-h exposure. Growth of P. subcapitata decreased with increasing clay percentage. For the treatment without shaking, the population of P. subcapitata cells for CK1 (medium control without sediment), CK2 (100% sand control, 1 g dry wt), 1.5% and 3% clay treatment increased to 18.3 × 104, 18.3 × 104, 18 × 104, and 17.5 × 104 cells/ml, respectively, which had no significant differences among them (p > 0.05). But the algal cell population for 6% clay treatment was significantly reduced to 14.6 × 104 cells/ml compared to the controls (p < 0.05). The population of P. subcapitata cells in the treatment with 29% clay only increased 6.2-fold after 72-h incubation. For the treatment with shaking, the population for 3% clay treatment was significantly reduced to 13.7 × 104 cells/ml when compared to the controls (p < 0.05). The population of P. subcapitata cells in the treatment with 29% clay only increased 3.6-fold after 72-h incubation. When clay content was no less than 3%, the shaking and nonshaking treatments had significant differences (p < 0.05).

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Figure 2. Populations of Pseudokirchneriella subcapitata after 72-h exposure to 1 g (dry wt) of mixed sediments with different clay contents with and without shaking of flasks during the assay. Error bars indicate standard deviations (n = 3). CK 1 is the culture media control without sediment, and CK 2 is 100% sand control. White circles represent the flasks incubated without shaking, and dark circles represent those with shaking twice per day by hand during the assay. The asterisk (*) indicates a statistically significant difference between shaking and without shaking treatment with the same clay content, and the letters (a, b, c, d, e, f, and g) indicate statistically significant differences between different clay content treatments (p < 0.05).

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Spiked sediment toxicity assay

Two different sediments, fine-grained sediment S1 (29% clay) and coarse-grained sediment S2 (2.9% clay), were used in the spiked sediment toxicity tests, with and without shaking the flasks during the incubation. The growth curves of P. subcapitata's cell density after 72-h exposure to the medium control (without sediments), S1, and S2 under incubation conditions are shown in Figure 3. After 72-h exposure, the population of P. subcapitata cells in the sediment control without shaking increased 6.2-fold (S1) and 17.3-fold (S2), which were higher than 3.9-fold (S1) and 12-fold (S2) found for the treatment with shaking. Shaking and nonshaking treatments for 72 h had significant differences (p < 0.05). The measured pH values ranged from 6.89 to 7.74 during the 72-h exposure, within the maximum 1.5 pH unit increase for test acceptability 27.

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Figure 3. Growth curves of cell density of Pseudokirchneriella subcapitata during 72-h exposure to 1 g (dry wt) fine sediment S1 (29% clay) and coarse sediment S2 (2.9% clay) under different incubation conditions. Error bars mean standard deviations (n = 3). The letters (a, b, c, and d) indicate statistically significant differences between treatments (p < 0.05) after 72-h exposure.

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Good sigmoidal-shaped dose–response curves (Fig. 4) were obtained for the sediments spiked with Cu and diuron. Growth of algae in beads was inhibited with increasing concentrations of Cu or diuron in the sediments. The EC50, no-observable-effect concentration, and lowest-observable-effect concentration values were determined for the two model compounds in the water and two sediments (Tables 3 and 4). The coefficients of variation (n = 2) of EC50 for Cu and diuron were between 1 and 14%, which showed good reproducibility. The results showed that the EC50 value of Cu or diuron in the sediment S1 was higher than that in sediment S2. This is attributable to a higher bioavailable toxicant (Cu or diuron) in the sediment S2. The sediments spiked with Cu or diuron showed higher toxicity to P. subcapitata in the treatments with shaking than in the treatment without shaking the flasks. This further demonstrated the effects of clay content and shaking on algal growth and thus algal toxicity to sediment.

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Figure 4. Cell density of Pseudokirchneriella subcapitata of dissolved beads exposed for 72 h to fine sediment S1 (29% clay) and coarse sediment S2 (2.9% clay) spiked with Cu and diuron. Error bars mean standard deviations (n = 3).

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Table 3. Effect concentrations of Cu or diuron solution, individual Cu or diuron spiked sediments including fine sediment (S1) and coarse sediment (S2), and four field sediments on the immobilized Pseudokirchneriella subcapitata growth inhibition
 Shaking methodEC50 (mean ± SD)NOEC (mean ± SD)LOEC (mean ± SD)
  1. EC50 = concentration of a target compound in spiked sediment or percentage of field sediment that inhibits growth yield by 50% (n = 2); SD = standard deviation; NOEC = no observed effect concentration (n = 2); LOEC = lowest observed effect concentration (n = 2).

Water only test (µg/L)  
 Cu 17.4 ± 2.06.0 ± 0.711.2 ± 1.1
 Diuron 22.3 ± 2.59.4 ± 0.719.5 ± 0.6
Sediment test
 Spiked sediment Cu (mg/kg)  
  S1 + CuWithout shaking1,189 ± 105372 ± 11761 ± 6
  S2 + CuWithout shaking142 ± 19203 ± 8372 ± 11
  S1 + CuShaking twice a day615 ± 846 ± 5103 ± 8
  S2 + CuShaking twice a day71 ± 921 ± 132 ± 14
  Diuron (mg/kg)  
  S1 + diuronWithout shaking1.16 ± 0.030.55 ± 0.211.00 ± 0.14
  S2 + diuronWithout shaking0.99 ± 0.130.15 ± 0.070.55 ± 0.21
  S1 + diuronShaking twice a day0.81 ± 0.080.15 ± 0.070.3 ± 0.14
  S2 + diuronShaking twice a day0.76 ± 0.060.15 ± 0.070.3 ± 0.14
 Field sediment Percentage (%)  
  AWithout shaking79.0--
  BWithout shaking41.4--
  CWithout shaking46.0--
  DWithout shaking76.2--
Table 4. Measured copper or diuron concentrations in spiked sediments and corresponding overlying water
Spiked sedimentMeasured concentrationa
  Overlying water
Nominal concentrationSpiked sedimentWithout shakingShaking twice a day
 Cu (mg/kg)Cu (µg/L)Cu (µg/L)
  • a

    The copper or diuron concentrations in the sediments and overlying water were mean concentrations on day 0 and day 3 of the bioassay.

S1 + Cu (mg/kg)
 12,00012,008190196
 6,0006,0589396
 3,0003,0092931
 1,5001,4421821
 7507561011
 375380<5<5
 188208<5<5
 94101<5<5
Control11<5<5
S2 + Cu (mg/kg)
 1,5001,512217219
 750769106108
 3753914445
 1881932524
 94971211
 4749<5<5
 2422<5<5
Control1<5<5
S1 + diuron (mg/kg)Diuron (mg/kg)Diuron (µg/L)Diuron (µg/L)
 5027.4487518
 2514.9269280
 12.58.4149132
 6.34.37875
 3.22.03531
 1.60.91616
 0.80.477
 0.40.122
Control000
S2 + diuron (mg/kg)
 207.7152160
 105.4107103
 52.65254
 2.51.22322
 10.61314
 0.50.354
 0.250.122
 Control000

Control sediment elutriates toxicity test

As the quality control and assurance, sediment controls were tested for the algal toxicity of their elutriates. The percentages of inhibition of the elutriates of control A, control B, control C, and control D were 0.3 ± 3.9, 0.7 ± 4.0, 1.2 ± 1.6, 1.5 ± 3.0%, respectively. No significant differences were found between them and the medium control (0 ± 4.9%) (p > 0.05).The results showed no toxicity to free algae P. subcapitata after 72-h exposure. Because corresponding sediments controls A, B, C, and D had the same physical properties with the test sediment samples A, B, C, and D, respectively, but showed no toxicity to free algae P. subcapitata, they could be used as the controls and diluents of the test sediments to obtain effective dilution series.

Field sediments toxicity assay and TIE

Physiochemical characteristics of the test sediment samples from the field sites are given in Table 1. An initial sediment toxicity test showed that all investigated sediment samples exhibited toxicity to the green alga P. subcapitata with 62.5% (C) to 85.9% (A) inhibition after 72-h exposure at 100% concentration, and EC50 values of the sediments A, B, C, and D was 79.0, 41.4, 46.0, and 76.2%, respectively (Table 3).

Further toxicity tests were performed to determine the toxic classes (ammonia, metals, and organics) in the four contaminated sediments. Toxicity identification evaluation manipulations were performed with zeolite, resin, and activated charcoal to remove toxics ammonia, metals, and organics in the four field sediment samples. As shown in Figure 5, adding zeolites had no significant effect on the algal growth, suggesting that ammonia played a minor role in the sediment toxicity. However, the percentage of growth inhibition of P. subcapitata was significantly reduced by adding cation exchange resin and activated charcoal, respectively, for both sediment B and C, whereas toxicities of sediments A and D were reduced remarkably only by adding activated charcoal. This suggested that metals and organics could be the suspect toxicants of sediment B and C, whereas organics were suspected as the major toxicants of sediments A and D.

thumbnail image

Figure 5. Toxicity identification evaluation for the four field sediment samples by various treatments with zeolite, resin, and activated charcoal in comparison with the baseline toxicity. Error bars mean standard deviations (n = 3).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. REFERENCES

The method for the whole-sediment toxicity test was developed by using alginate immobilized algae beads. After the test, the algae released from immobilized beads could be easily counted in a hemocytometer. Various key experimental factors such as stability of algal beads and algal cells growth in beads, influences of sediment particle size and shaking the flask during the test, and control or diluent sediment preparation were optimized. The developed method was successfully applied to the toxicity assays of the spiked sediments and four field sediment samples.

Stability of beads and growth of cells

Alginate bead stability is an important factor in sediment toxicity tests. Alginate matrix is susceptible to cation chelating agents (e.g., phosphate and citrate) and antigelling cations (e.g., sodium and magnesium), which can lead to disrupting the bead structure 20. The stability of beads also can depend on the immobilized algal species 28. In the present study, beads prepared using 4% alginate solution and hardened with 4% CaCl2 · 2H2O solution were found to be stable and suitable to allow a good growth of P. subcapitata cells by an approximately 18.5-fold increase after 72-h exposure (Table 2). Although the immobilized algal beads showed slower growth than free cells as demonstrated in the present study and a previous study 29, growth of algae cells in the beads could meet the criteria of at least a 16-fold increase in cell number over 72 h for test acceptability 27. For the sediment toxicity test, fitting a 16-fold benchmark is difficult because sediment particles affect the growth of algae, and as a result the benchmark would need adjusting.

An advantage to using immobilized algal beads in a whole-sediment toxicity test is that this overcomes the difficulty in cell counting that arises when using free cells. In a whole-sediment toxicity test using immobilized beads, one can easily distinguish immobilized algal cells from sediment particles. After exposure, algal cells dissolved from the beads could be easily counted by a light microscope. Cell density in beads was satisfactorily determined by hemocytometer counting. Cellular densities lower than 104 cells/ml cannot be counted by a hemocytometer. In the present study, however, after a 72-h exposure test, 10 beads from each flask were concentrated by dissolving in sodium citrate to a final volume of 2 ml, and algal cells were counted accurately. Although hemocytometer counting is undoubtedly a labor-intensive method, obtaining accurate data for those laboratories without flow cytometry is quite economical and practical.

Effects of clay fraction and shaking

Sediment S1 with a high clay content was a natural sediment collected from a reservoir. S1 was found not to be toxic to P. subcapitata, although contaminants (metals, polycyclic aromatic hydrocarbons, and pesticides) were present in S1 (Table 1), suggesting that these contaminants were biologically unavailable. First, the overlying water of S1 in the toxicity test was analyzed, and no target contaminants (metals, polycyclic aromatic hydrocarbons, and pesticides) were detected. Second, S1 was extracted three times with 0.01M CaCl2 solution, acetone, and dichloromethane to remove the bioavailable toxicants (extractable metals, polycyclic aromatic hydrocarbons, and pesticides). No significant differences were found in algal toxicity between untreated S1 and extracted S1 in the sediment toxicity test. The high binding capacity of contaminants to clay fractions of sediment S1 reduced their bioavailability and thus showed no toxicity difference between extracted and untreated sediment S1. Thus, sediment S1 was considered as a clean sediment in the spiking experiment.

The present study (Fig. 2 and Fig. 3) showed that growth of P. subcapitata in alginate beads decreased with an increasing proportion of clay fraction in sediment. This may be explained by the covering of the clay fraction (<0.002 mm) on the algae-alginate beads, thus inhibiting algal photosynthesis 6.

Figure 4 shows very different responses to Cu and diuron in the spiked sediment toxicity test (Fig. 4). First, the effect of shaking was different; there were large differences for Cu and small differences for diuron. Second, the toxicities of the two sediments were different. S2 was much more toxic than S1 for Cu, but lower differences were found between the two sediments spiked with diuron. The toxicity test of the sediments spiked with Cu and diuron demonstrated that growth of P. subcapitata is primarily affected by dissolved Cu or diuron in the overlying water rather than by sediment-associated Cu or diuron. Copper can be easily adsorbed onto sediment, whereas diuron has a low KOC (2.6), indicating a relatively low tendency to sorb onto sediments. During the assay, shaking the flasks could make algal beads and sediment contact sufficiently but artificially increased their toxicity compared with those without shaking (Table 3) because of the covering of clays on the surfaces of the alginate beads and reduction of light penetration in the media. Therefore, shaking the flasks during the incubation could significantly reduce the growth of cells. In addition, sediments in real aquatic environments would not be disturbed in most cases, as in the situation of flask shaking. Incubation of test media without shaking would be preferred in the whole-sediment toxicity test, and in fact it is also more environmentally realistic.

Species sensitivity

Previous studies showed different sediment toxicities of the reference toxicant Cu to various species 6, 30–32. However, no toxicity data have been reported regarding diuron in sediments. Marine benthic diatom (Cylindrotheca closterium) showed high sensitivity, and the EC50 value was reported to be 26 mg/kg Cu spiked in natural marine sand 6, which is lower than the EC50 values from the present study for coarse-grained sediments (142 mg Cu/kg) and fine-grained sediment (1,189 mg Cu/kg). Previous studies also reported some lethal toxicity data such as median lethal concentration values of Cu-spiked sediments to Tubifex tubifex ranging from 88 to 106 mg/kg 30, median lethal concentration of 1,310 mg Cu/kg for adult and 790 mg Cu/kg for juvenile amphipod Melita plumulosa31, and median lethal concentration values of 426 mg Cu/kg for Chironomus riparius larvae after 10 d of exposure to Cu-spiked sediments 32. The present study and previous studies suggest that different species have variable sensitivity toward toxicants in sediments, and their toxicity can be influenced by toxicants and sediment components.

Field sediment toxicity assay

Diluent sediment and control sediment were crucial in the real sediment toxicity test. As found in the present study, the physicochemical properties and particle size distribution could affect algal toxicity of a sediment. Diluent sediment should have similar properties to the test sediment, and concentrations of contaminants should be under background levels 21. Control sediment of a similar composition to the test sediment is perhaps the most environmentally realistic diluent, particularly if it originates from the same ecosystem 8. No choice is more appropriate than using the test sediment after its potential toxics were removed by extraction as control/diluent. A control or diluent sediment preparation method was developed by using a series of extractions with 0.01M CaCl2 solution, acetone, and dichloromethane. Removing potential toxics in the control sediment was confirmed by an algal toxicity test to the elutriate of the control sediment. In the present study, we successfully applied the control or diluent that originated from the four field sediment samples in the whole sediment toxicity test.

In addition to baseline toxicity tests for the four sediments from the polluted rivers in the Pearl River Delta region, a phase I TIE approach was also applied to identify toxic classes in the sediments (Fig. 5). Sediments B and C were more toxic in the other two sediments A and D. As shown in Table 1, sediments B and C had much higher metal concentrations. Manipulation of TIE (Fig. 5) also showed that adding metal-absorbing resin into the sediments significantly reduced the algal toxicity, suggesting that metals were a major toxic class in the two sediments (B and C). Adding activated charcoal significantly reduced the algal toxicity of the four sediments (Fig. 5), suggesting that organic contaminants were the major toxic class in the four sediments. Based on the chemical analysis data (Table 1), polycyclic aromatic hydrocarbons carbofuran, cyanazine, and cyfluthrin at high concentrations in the four sediments most probably contributed a major part of the toxicity.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. REFERENCES

A growth inhibition test using immobilized freshwater microalgae P. subcapitata was developed to determine the whole-sediment toxicity. Various influencing factors such as bead preparation and stability, shaking, sediment particle size distribution, and control/diluent sediment selection were optimized for the sediment toxicity test. Immobilized algae could be used in toxicity assay of whole sediments, including fine and coarse sediments. Control/diluent sediment could be prepared from the corresponding field sediment by repeated extractions with 0.01 M CaCl2 solution and acetone and dichloromethane to remove potential toxic contaminants.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. REFERENCES

The authors acknowledge financial support from the National Natural Science Foundation of China (NSFC U1133005, 20977092, 40821003, and 40688001). We also acknowledge the partial financial support received from the Earmarked Fund from the State Key Laboratory of Organic Geochemistry (SKLOG2009A02) and the National Water Research Project (2009ZX07528-001). Thanks also to M. Adams and J. Stauber (CSIRO, Australia) for their critical review and useful comments on the manuscript. This is Contribution No. 1413 from the Guangzhou Institute of Geochemistry, Chinese Academy of Sciences.

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  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. REFERENCES
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