ARTICLE pubs.acs.org/est
Effects of Cd, Cu, Ni, and Zn on Brown Tide Alga Aureococcus Anophagefferens Growth and Metal Accumulation Bin Wang,† Lisa Axe,† Zoi-Heleni Michalopoulou,‡ and Liping Wei*,§ †
Civil and Environmental Engineering, ‡Mathematical Sciences, and §Chemistry and Environmental Science, New Jersey Institute of Technology, University Heights, Newark, New Jersey 07102, United States
bS Supporting Information ABSTRACT: Trace metals play important roles in regulating phytoplankton growth and could influence algal bloom development. Laboratory studies were conducted to evaluate the influence of environmentally relevant concentrations of Cd, Cu, Ni, and Zn on Aureococcus anophagefferens bloom (brown tide) development. Results show that the elevated Ni2+ concentrations, e.g. those of brown tide waters in the northeastern US, greatly stimulated A. anophagefferens growth (as compared to the control without Ni addition), yet, only low amounts of dissolved Ni were sequestered, thus leaving excessive Ni directly promoting A. anophagefferens blooms. The medium effective concentration EC50 (Me2+) suggests A. anophagefferens has similar Cd sensitivity but much greater Cu tolerance as compared to cyanobacteria, as such, excessive Cu could indirectly promote A. anophagefferens blooms by inhibiting competitors such as Synechococcus sp. The effects of Ni and Cu promoting growth are consistent with the recent genomic study of this alga. In addition, Zn2+ concentrations lower than those in brown tide waters enhance A. anophagefferens growth, but Zn sequestration in A. anophagefferens would not substantially reduce total dissolved Zn in these waters. Overall, this study, showing that excessive Cu and Ni likely promote brown tides, provides evidence for trace metal linkages in algal bloom development.
’ INTRODUCTION Brown tide blooms caused by the pelagophyte Aureococcus anophagefferens have been recurring in the New York/New Jersey coastal area since the first observations in 1985.1,2 Sunda et al. 3 referred to these blooms as ecosystem disruptive algal blooms (EDABs) because they may significantly change or degrade ecosystem function. For example, A. anophagefferens blooms increase water column light attenuation and discolor the water.2 Furthermore, brown tides cause feeding secession in zooplankton 4 and shellfish species,5 and eventually alter food web structure.2,6 Trace metals, particularly Fe, Mn, Cu, Zn, Co, Ni, and even the nonessential metal Cd, play a key role in marine phytoplankton carbon, nitrogen, and phosphorus acquisition.7 In coastal waters several of these metals, such as Cd, Cu, Ni, and Zn, are common contaminants from industrial wastewater and sewage discharges, antifouling paints, fossil fuel combustion, and urban and agricultural runoff.8 A number of coastal algal blooms were found to have trace metal links. Iron was involved in the occurrence of harmful algal blooms of dinoflagellate Gymnodinium breve in southern Florida,9 cobalt has been implicated in the incidence of the blooms of prymnesiophyte Chrysochromulina polylepis at Kattegat, southeast Sweden,10 and selenium has also been identified as a potentially important factor in Gymnodinium nagasakiense dinoflagellate blooms in Tanabe Bay, Japan.11 Some of the nonessential trace metals such as Cd may also promote algal growth by substituting for nutrients when essential metals are depleted, as observed when Cd addition resulted in a Zn-deficient r 2011 American Chemical Society
diatom Thalassiosira weissflogii that grew at 90% of its maximum growth rate.12 The occurrence of algal blooms, particularly in shallow coastal waters, could affect trace metal speciation and biogeochemical cycling.13 Development of diatom and dinoflagellate blooms in a subestuary of the Chesapeake Bay rapidly reduced arsenate to arsenite and methylated species, and increased organically complexed copper from 2040% to 60100% of the total dissolved copper.14 Diatom blooms in the southern portion of the San Francisco Bay reduced concentrations of dissolved Zn, Cd, and Ni by as much as 75% of their prebloom concentrations, while dissolved Cu increased 20%.15 Apparently the phytoplankton assemblage and the productivity greatly affect total dissolved and bioavailable metal concentrations. The significant capability of brown tide bloom alga A. anophagefferens to utilize organic nitrogen as a sole nitrogen source (comparable to heterotrophic bacteria) was identified as important in algal physiology for bloom initiation, especially when the inorganic nitrogen levels were relatively low for the competing algal species.2 A. anophagefferens can also grow heterotrophically using organic carbon as the sole carbon source.16,17 Organic nitrogen and carbon utilization are likely supported by a suite Received: August 10, 2011 Accepted: November 21, 2011 Revised: November 8, 2011 Published: November 21, 2011 517
dx.doi.org/10.1021/es202790p | Environ. Sci. Technol. 2012, 46, 517–524
Environmental Science & Technology
ARTICLE
Table 1. Metal Concentrations of Cd, Cu, Ni, and Zn in Experiments and Coastal Waters Where Brown Tides Were Observeda metal concentrations metal
control
metal concentrations in
in growth rate study
total EDTA
metal concentrations
metal accumulation study (nM) in coastal waters (nM)
Cd [CdT] N/Ab 0
30 nM
0.3 μM
3 μM
30 μM
[Cd ] N/A
0.18 nM 1.8nM
17.6 nM 176 nM
pCd
11.33
10.33
9.33
1.2 μM
12 μM
120 μM 120 μM
8.8 pM 12.03
N/A
100 μM + [CdT]
8.33
0.656.5
0.0251.6 c
0.656.5
0.050.22 d
pCd 10.769.76
11.711.2 d
Cu [CuT] 20 nM 0
[Cu ] 0.15pM pCu 13.8
10 μM + [CuT] (pCu = 9.03)
551 c
88.1 pM 0.88 nM 8.8 nM 11.03 10.03 9.03
100 μM + [CuT] (pCu e 10.03)
0.0030.025 d 13.711.7 d
3 μM
30 μM
30 μM
10 μM + [NiT] (pNi e 9.23)
0.16155
190.8 c
100 μM + [NiT] (pNi g 10.23)
0.16155
0.564 e
pNi 107
9.57.4 f
Ni [NiT] N/A
300 μM
0
[Ni ]
N/A
9.5 pM
95 pM
0.95 nM 9.5 nM
pNi
N/A
11.23
10.23
9.23
8.23
6 nM
60 nM
0.6 μM
6 μM
Zn [ZnT] 80 nM [Zn0 ] pZn
47.2 pM 3.5 pM 10.7 11.8
60 μM
35.4 pM 0.35 nM 3.54 nM 35.4 nM 100 μM + [ZnT] 10.8 9.8 8.8 7.8
6.4, 64
572 c
6.4, 64 pZn 8.57, 7.57
0.626.6 d 9.57.9 d
[MeT] = total metal concentration, [Me0 ] = total inorganic metal concentration ([Cd0 ] = [Cd2+] + [CdOHCl(aq)] + [CdCl+] + [CdCl3] + [CdCl2(aq)]; [Cu0 ] = [Cu2+] + [CuOH+] + [Cu(OH)2(aq)] + [CuCl+] + [CuCO3(aq)] + [Cu(CO3)22] + [CuSO4(aq)]; [Ni0 ] = [Ni2+] + [NiHCO3+] + [NiCl+] + [NiCO3(aq)] + [NiSO4(aq)]; [Zn0 ] = [Zn2+] + [ZnOHCl(aq)] + [ZnCl3] + [ZnCl+] + [ZnCl2(aq)] + [ZnCO3(aq)] + [ZnSO4(aq)].), and pMe = log [Me2+]. b Not added. c Breuer et al.,29 Clark et al.,30 Kozelka and Bruland,31 and Wells et al.32 d Kozelka and Bruland.31 e [Ni0 ] = 5070% of [NiT] according to Slaveykova et al.49 f [Ni2+] is estimated by MINEQL 23 based on 0.564 nM Ni0 in synthetic ocean water.21 a
of metalloenzymes and trace metal cofactors.7,18 A recent genomic study has shown that A. anophagefferens contains significantly more genes encoding Se, Mo, Ni, and Cu requiring enzymes than other phytoplankton and that these enzyme may facilitate growth and organic matter utilization in this alga.19 The role of trace metals on A. anophagefferens growth and metal sequestration has been studied only with Fe, where it was found that A. anophagefferens required low levels for growth (a 5 nM quota of iron was needed for a bloom of 106 cells/mL).20 Yet, the role of other trace metals on the brown tide bloom promotion and metal sequestration has not been studied to date. This work focuses on the influence of trace metals, Cd, Cu, Ni, and Zn, on A. anophagefferens growth and metal sequestration. In growth rate experiments, the effects of metals on A. anophagefferens growth were evaluated after approximately 18 generations of exposure to a range of Cd2+, Cu2+, Ni2+, and Zn2+ concentrations. In metal accumulation experiments, sequestration was investigated by measuring adsorbed and intracellular metal concentrations using radioisotopes 109Cd, 63Ni, and 65Zn. Results provide insights on trace metal regimes that promote or inhibit A. anophagefferens blooms.
carbonate bottles were used because they have very low metal contamination and are known to minimally adsorb metals from media.22 Media and cultures were handled in a HEPA filtered laminar flow hood. The culture media Aquil-Si were prepared following Price et al.21 Briefly, Chelex-100-treated and microwave sterilized synthetic ocean water (SOW) was supplemented with 0.2 μm-filtersterilized nitrate, phosphate, vitamin mixture, and trace metal mixture (containing Fe, Mn, Co, Cu, Zn, and ethylenediaminetetraacetic acid (EDTA)). In control media (Aquil-Si) Cu and Zn ion levels were pCu 13.8 and pZn 10.7 (pMe = log [Me2+]), and Cd and Ni were not added. Metal speciation was calculated using the thermodynamic equilibrium model MINEQL23 with an updated database from Schecher.24 The media for the growth rate study were prepared by adding equimolar Cd-EDTA, Cu-EDTA, Ni-EDTA, or Zn-EDTA solutions to Aquil-Si containing either 10 or 100 μM EDTA to control free ion concentrations (Table 1). The resulting media EDTA was less than 500 μM (as recommended for phytoplankton culturing21), and the free metal ion concentrations [Me2+] covered most of the [Me2+] of northeastern U.S. coastal waters with reported or recurring brown tides (i.e., brown tide waters, Table 1). To prepare media for metal accumulation experiments, radioisotopes were used to tag the nonradioactive forms, 109Cd (6.1061 kBq L1 in 0.5 M HCl), 63Ni (3.53354 kBq L1 in 0.5 M HCl), and 65Zn (3.6236.2 kBq L1 in 0.5 M HCl) (all from Eckert & Ziegler Isotope Products) in EDTA-free Aquil-Si (i.e., no trace metal mixture and EDTA addition). Ni concentrations in media were further elevated by adding nonradioactive Ni2SO4 (Alfa Aesar, >99.97% purity). A Cu radioisotope was not obtained for this study. After adding the metals and adjusting pH to 8.1 ( 0.1, the culture media were equilibrated for at least 24 h. The resulting total metal concentrations ranged from 6.51 1010 to 6.50 109 M for Cd, 1.64 1010 to 1.55 107 M
’ MATERIALS AND METHODS Algal Culturing and Media Preparation. Axenic Aureococcus anophagefferens (CCMP 1984) was maintained in artificial seawater media Aquil-Si (Aquil without silicate addition) 21 with a pH of 8.1 ( 0.1 in a Thermo Scientific 818 Precision diurnal growth chamber with 12 h: 12 h light: dark cycle, 120 μmol photons 3 m2 3 s1 illumination at 19 ( 0.2 °C. The culture was grown in 125 or 250 mL polycarbonate bottles (Nalgene) which were cleaned following the procedure of Price et al.:21 a detergent soak, tap water rinse, deionized water rinse, 24 h 1 N HCl soak, and a final (ample) Milli-Q water (18.2 MΩ 3 cm) rinse. Poly518
dx.doi.org/10.1021/es202790p |Environ. Sci. Technol. 2012, 46, 517–524
Environmental Science & Technology for Ni, and 6.43 109 to 6.43 108 M for Zn, with free metal ion concentrations of pCd 10.76, 10.26, and 9.76; pNi 10, 9, 8, and 7; and, pZn 8.57 and 7.57 (Table 1). Quantifying the Effects of Cd, Cu, Ni, and Zn Exposure on Aureococcus anophagefferens Growth. The experimental procedure was similar to that in Ahner et al. 25 and Wei et al.26 A. anophagefferens was inoculated to the control media (Aquil-Si) and the metal amended media (Aquil-Si with different initial Cd2+, Cu2+, Ni2+, or Zn2+ concentrations) in 125 mL polycarbonate bottles. Once the culture reached late exponential growth phase, an aliquot was withdrawn and inoculated to fresh media at 1:100 v:v ratio to continue the metal exposure and acclimation (i.e., subculturing). After acclimation for approximately 18 generations (25 days), A. anophagefferens was further subcultured (∼1 to 100 mL fresh media) and the in vivo fluorescence (Turner Designs Trilogy fluorometer) over the exponential growth phase (38 days, Supporting Information) was used to obtain the growth rate, μ (d1): ln Xt = μ 3 t + ln X0, where Xt and X0 are in vivo fluorescence (RFU, relative fluorescence unit) of culture at time t (d) and time 0 (initially), respectively. Since a linear relationship between in vivo fluorescence and cell density was previously observed for A. anophagefferens at in vivo fluorescence
