Jennifer L. Stauber

GROWTH-INHIBITING EFFECTS OF 12 ANTIBACTERIAL AGENTS AND THEIR MIXTURES ON THE FRESHWATER MICROALGA PSEUDOKIRCHNERIELLA SUBCAPITATA

The growth-inhibiting and binary joint effects of 12 antibacterial agents on the freshwater green alga Pseudokirchneriella subcapitata (Korschikov) Hindák were investigated over 72-h exposures. The toxicity values (the median inhibitory concentration value, in micromoles) in decreasing order of sensitivity were triclosan (0.0018)>triclocarban (0.054)>roxithromycin (0.056)>clarithromycin (0.062)>tylosin (0.20)>tetracycline (2.25)>chlortetracycline (3.49)>norfloxacin (5.64)>sulfamethoxazole (7.50)>ciprofloxacin (20.22)>sulfamethazine (31.26)>trimethoprim (137.78). Several of these antibacterial compounds would be toxic at the micrograms per liter concentrations reported in surface waters and sewage effluents. Simple additive effects were observed in binary mixtures of sulfonamides, and most tylosin, triclosan, or triclocarban combinations. Potentially synergistic effects were observed in binary mixtures of the same class, such as macrolides, tetracyclines, and fluoroquinolones, as well as in some combined drugs, such as trimethoprim and sulfonamides or tylosin and tetracyclines. Potentially antagonistic effects were only observed between tylosin and triclocarban, triclosan and norfloxacin, and triclocarban and norfloxacin. Although present at low concentrations in the aquatic environment, mixtures of these antibacterial agents can potentially affect algal growth in freshwater systems due to their combined action.

pH-dependent toxicity of copper and uranium to a tropical freshwater alga (Chlorella sp.).

Copper (Cu) and uranium (U) are of potential ecotoxicological concern to tropical freshwater organisms in northern Australia as a result of mining activity. No local data on the toxicity of these metals to tropical freshwater algae are currently available. The aim of this study was to investigate the effect of pH (5.7 and 6.5) on the toxicity of Cu and U to the green alga Chlorella sp. in a synthetic softwater representative of fresh surface waters in sandy-streams of tropical northern Australia. The effects of Cu and U on algal growth (cell division) rate after a 72-h exposure were determined. Intracellular and extracellular (membrane-bound) metal concentrations at the two selected pH values were also compared. Based on the 72-h minimum detectable effect concentrations (MDEC), Chlorella sp. was approximately 20-fold more sensitive to Cu (0.7 and 1.4 µg l(-1) at pH 6.5 and 5.7, respectively) than U (13 and 34 µg l(-1) at pH 6.5 and 5.7, respectively), and more sensitive than other Australian tropical freshwater organisms. The toxicity of Cu and U was highly pH-dependent. Copper concentrations required to inhibit growth (cell division) rate by 50% (72-h EC(50)) increased from 1.5 to 35 µg l(-1) as the pH decreased from 6.5 to 5.7. Similarly, the 72-h EC(50) values for U increased from 44 to 78 µg l(-1) over the same pH range. Calculation of Cu and U speciation using the geochemical model HARPHRQ, showed that differences in the concentrations of the free metal ions (Cu(2+) and UO(2)(2+)) were only minimal (<10%) between pH 5.7 and 6.5. The decreased toxicity at pH 5.7 was due to lower concentrations of cell-bound and intracellular Cu and U compared to those at pH 6.5. These results are explained in terms of the possible mechanism of competition between H(+) and the metal ion at the cell surface.

Speciation and Bioavailability of Trace Metals in Water: Progress Since 1982

The advances in studies of trace metal speciation and bioavailability since Mark Florence’s 1982 review of the topic, published in Talanta, have been comprehensively reviewed. While the relative merits of kinetic and equilibrium approaches are still being determined, advances in the applications of stripping voltammetry, including the application of microelectrodes and an appreciation of detection windows in both CSV and ASV, have been matched by the introduction of new dynamic techniques including diffusive gradients in thin films (DGTs), permeation liquid membranes (PLMs), and improved applications of chelating resins. There have also been improvements in equilibrium techniques such as ion-selective electrodes and Donnan dialysis. The ability of geochemical speciation models to predict metal complexation by natural organic matter has greatly improved, yet the models still require validation against field measurements. More reliable and relevant bioassays have been developed using sensitive species such as algae and bacteria, and improved protocols are eliminating uncertainties particularly due to problems with high cell densities, and are allowing more useful comparisons with chemically estimated bioavailability. While the free-ion activity model has provided an improved interpretation of the relative toxicities observed with different metal species, its extension to the biotic ligand model is leading to better predictions of acute effects at least on higher organisms. The extension of such approaches to studies of chronic effects at ‘natural’ concentrations using unicellular organisms remains a challenge, as does the evaluation of whether such approaches are simplified limiting cases of a more dynamic natural situation where chemical reactivity and rates of metal transport could be important.

Toxicity, biotransformation, and mode of action of arsenic in two freshwater microalgae (Chlorella sp. and Monoraphidium arcuatum)

The toxicity of As(V) and As(III) to two axenic tropical freshwater microalgae, Chlorella sp. and Monoraphidium arcuatum, was determined using 72-h growth rate-inhibition bioassays. Both organisms were tolerant to As(III) (72-h concentration to cause 50% inhibition of growth rate [IC50], of 25 and 15 mg As[III]/L, respectively). Chlorella sp. also was tolerant to As(V) with no effect on growth rate over 72 h at concentrations up to 0.8 mg/L (72-h IC50 of 25 mg As[V]/L). Monoraphidium arcuatum was more sensitive to As(V) (72-h IC50 of 0.25 mg As[V]/L). An increase in phosphate in the growth medium (0.15-1.5 mg PO4(3-)/L) decreased toxicity, i.e., the 72-h IC50 value for M. arcuatum increased from 0.25 mg As(V)/L to 4.5 mg As(V)/L, while extracellular As and intracellular As decreased, indicating competition between arsenate and phosphate for cellular uptake. Both microalgae reduced As(V) to As(III) in the cell, with further biological transformation to methylated species (monomethyl arsonic acid and dimethyl arsinic acid) and phosphate arsenoriboside. Less than 0.01% of added As(V) was incorporated into algal cells, suggesting that bioaccumulation and subsequent methylation was not the primary mode of detoxification. When exposed to As(V), both species reduced As(V) to As(III); however, only M. arcuatum excreted As(III) into solution. Intracellular arsenic reduction may be coupled to thiol oxidation in both species. Arsenic toxicity most likely was due to arsenite accumulation in the cell, when the ability to excrete and/or methylate arsenite was overwhelmed at high arsenic concentrations. Arsenite may bind to intracellular thiols, such as glutathione, potentially disrupting the ratio of reduced to oxidized glutathione and, consequently, inhibiting cell division.

Uptake and internalisation of copper by three marine microalgae: comparison of copper-sensitive and copper-tolerant species

Although it has been well established that different species of marine algae have different sensitivities to metals, our understanding of the physiological and biochemical basis for these differences is limited. This study investigated copper adsorption and internalisation in three algal species with differing sensitivities to copper. The diatom Phaeodactylum tricornutum was particularly sensitive to copper, with a 72-h IC50 (concentration of copper to inhibit growth rate by 50%) of 8.0 microg Cu L(-1), compared to the green algae Tetraselmis sp. (72-h IC50 47 microg Cu L(-1)) and Dunaliella tertiolecta (72-h IC50 530 microg Cu L(-1)). At these IC50 concentrations, Tetraselmis sp. had much higher intracellular copper (1.97+/-0.01 x 10(-13)g Cu cell(-1)) than P. tricornutum (0.23+/-0.19 x 10(-13)g Cu cell(-1)) and D. tertiolecta (0.59+/-0.05 x 10(-13)g Cu cell(-1)), suggesting that Tetraselmis sp. effectively detoxifies copper within the cell. By contrast, at the same external copper concentration (50 microg L(-1)), D. tertiolecta appears to better exclude copper than Tetraselmis sp. by having a slower copper internalisation rate and lower internal copper concentrations at equivalent extracellular concentrations. The results suggest that the use of internal copper concentrations and net uptake rates alone cannot explain differences in species-sensitivity for different algal species. Model prediction of copper toxicity to marine biota and understanding fundamental differences in species-sensitivity will require, not just an understanding of water quality parameters and copper-cell binding, but also further knowledge of cellular detoxification mechanisms.

Applications of flow cytometry to ecotoxicity testing using microalgae

Flow cytometry is a rapid method for the quantitative measurement of light scattering and fluorescent properties of cells. Although this technique has been widely applied to biomedical and environmental studies, its potential as a tool in ecotoxicological studies has not yet been fully exploited. This article describes the application of flow cytometry to the development of bioassays with marine and freshwater algae for assessing the bioavailability of contaminants in waters and sediments.

The effect of water hardness on the toxicity of uranium to a tropical freshwater alga Chlorella sp.

Uranium (U) derived from mining activities is of potential ecotoxicological concern to freshwater biota in tropical northern Australia. Few data are available on the effects of water hardness (Ca and/or Mg), which is elevated in U mine wastewaters, on the toxicity and bioavailability of U to freshwater biota, particularly algae. This study determined the effect of water hardness (8, 40, 100 and 400 mg CaCO(3) x l(-1), added as calcium (Ca) and magnesium (Mg) sulphate) on the toxicity (72 h growth rate inhibition) of U to the unicellular green alga, Chlorella sp., in synthetic freshwater, at constant pH (7.0) and alkalinity (8 mg CaCO(3) x l(-1)), similar in chemical composition to sandy coastal streams in tropical northern Australia. A 50-fold increase in water hardness resulted in a 5-fold decrease (P<or =0.05) in the toxicity of U to Chlorella sp. (i.e. the 72 h EC(50) increased from 56 to 270 micro g U l(-1)). Possible explanation for the ameliorative effect of water hardness includes: (i) competition between U and Ca and/or Mg for binding sites on the cell surface; and (ii) a change in U speciation, and hence, bioavailability. Results showed that extracellular (cell-surface) and intracellular U concentrations significantly (P<0.05) decreased (2-5-fold) as water hardness increased from 8 to 400 mg CaCO(3)x l(-1). Calculation of U speciation using the geochemical model HARPHRQ showed that there were no significant (P>0.05) differences in the predicted speciation (% distribution) of U amongst the four water hardness levels. The reduction in U toxicity with increasing water hardness was most likely due to competition between U and Ca and/or Mg for binding sites on the algal cell surface. The minimum detectable effect concentrations of U were approximately 3 and 24 times higher (at 8 and 400 mg CaCO(3)x l(-1) hardness, respectively) than the national interim U guideline value (0.5 micro g x l(-1)) for protecting aquatic ecosystems. Overall, the results reinforce the need for a more flexible U guideline based on a hardness-dependent algorithm, which may allow environmental managers to relax the national guideline for U on a site-specific basis.

ECOLOGICAL RISK ASSESSMENT IN THE CONTEXT OF GLOBAL CLIMATE CHANGE

Changes to sources, stressors, habitats, and geographic ranges; toxicological effects; end points; and uncertainty estimation require significant changes in the implementation of ecological risk assessment (ERA). Because of the lack of analog systems and circumstances in historically studied sites, there is a likelihood of type III error. As a first step, the authors propose a decision key to aid managers and risk assessors in determining when and to what extent climate change should be incorporated. Next, when global climate change is an important factor, the authors recommend seven critical changes to ERA. First, develop conceptual cause–effect diagrams that consider relevant management decisions as well as appropriate spatial and temporal scales to include both direct and indirect effects of climate change and the stressor of management interest. Second, develop assessment end points that are expressed as ecosystem services. Third, evaluate multiple stressors and nonlinear responses—include the chemicals and the stressors related to climate change. Fourth, estimate how climate change will affect or modify management options as the impacts become manifest. Fifth, consider the direction and rate of change relative to management objectives, recognizing that both positive and negative outcomes can occur. Sixth, determine the major drivers of uncertainty, estimating and bounding stochastic uncertainty spatially, temporally, and progressively. Seventh, plan for adaptive management to account for changing environmental conditions and consequent changes to ecosystem services. Good communication is essential for making risk-related information understandable and useful for managers and stakeholders to implement a successful risk-assessment and decision-making process. Environ. Toxicol. Chem. 2013;32:79–92.

Development of a whole‐sediment toxicity test using a benthic marine microalga

An acute whole-sediment toxicity test with a benthic marine microalga was developed and optimized using flow cytometry to distinguish algae (based on their chlorophyll a autofluorescence) from sediment particles. Of seven benthic marine algae screened, the diatom Entomoneis cf punctulata was most suitable because of its tolerance of a wide range of water and sediment physicochemical parameters, including salinity, pH, ammonia, and sulfide. A whole-sediment and water-only toxicity test based on inhibition of esterase activity in this species was developed. Enzyme activity rather than growth was used as the test endpoint, as nutrient release from sediments has previously been found to stimulate algal growth, potentially masking contaminant toxicity. The sensitivity of the bioassay to a range of metals (copper, zinc, cadmium, lead, arsenic, manganese) and phenol in water-only exposures was compared to the standard 72-h growth rate inhibition test. The esterase enzyme inhibition test was sensitive to copper, with a 3-h inhibitory concentration to cause a 50% (IC50) reduction in a fluorescein diacetate fluorescence value of 97 +/- 39 microg Cu/L. A concentration-dependent response was also observed in the presence of sediment particles (copper tailings), with and without dilution, using a control clean sediment. The primary route of exposure to copper was via pore water rather than by direct contact with tailings particles. This is the first whole-sediment bioassay developed with a benthic alga suitable for sediment quality assessment in marine/estuarine systems, and its advantages and limitations are discussed.

Development of multispecies algal bioassays using flow cytometry

Multispecies algal bioassays, suitable for assessing copper toxicity, were developed with three marine (Micromonas pusilla, Phaeodactylum tricornutum, and Heterocapsa niei) and three freshwater (Microcystis aeruginosa, Pseudokirchneriella subcapitata, and Trachelomonas sp.) microalgae. Flow cytometry was used to separate and count algal signals based on pigment fluorescence and cell size. Species were mixed together on the basis of equivalent surface areas to avoid the confounding effect on toxicity of increased biomass for metal binding. Under control conditions (no added copper), M. pusilla growth was inhibited in the presence of the other marine microalgae compared to single-species tests, while the opposite was true (i.e., growth stimulation) for M. aeruginosa and P. subcapitata in freshwater mixtures. Competition for nutrients, including CO2, and algal exudate production may account for these effects. Interactions between microalgal species also had a significant effect on copper toxicity to some species. In freshwater multispecies bioassays, the toxicity of copper to Trachelomonas sp. was greater in the presence of other species, with copper concentrations required to inhibit growth (cell division) rate by 50% (72-h [IC50]) decreasing from 9.8 to 2.8 microg Cu/L in single- and multispecies bioassays, respectively. In contrast, in marine multispecies bioassays, copper toxicity to the marine diatom P. tricornutum was reduced compared to single-species bioassays, with an increase in the 72-h IC50 value from 13 to 24 microg Cu/L. This reduction in copper toxicity was not explained by differences in the copper complexing capacity in solution (as a result of exudate production) because labile copper, measured by anodic stripping voltammetry, was similar for P. tricornutum alone and in the mixture. These results demonstrate that single-species bioassays may over- or underestimate metal toxicity in natural waters.