Merrin S. Adams

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.

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.

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.

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.

Dietary ingestion of fine sediments and microalgae represent the dominant route of exposure and metal accumulation for Sydney rock oyster (Saccostrea glomerata): A biokinetic model for zinc.

Past studies disagree on the extent to which dissolved or dietary uptake contribute to metal bioaccumulation in the filter-feeding Sydney rock oyster (Saccostrea glomerata) in urbanized estuaries. Although most data support the assumption that fine sediments are a major route of metal uptake in these bivalves, some studies based in the Sydney estuary, Australia, have indicated a poor correlation. In the present study, seawater, sediment and microalgae were radiolabelled with (65)Zn tracer and exposed to S. glomerata to assess the influence of dissolved and dietary sources to Zn bioaccumulation. Oysters in the dissolved-phase uptake experiment (5, 25 and 50 μg L(-1) (65)Zn for 4 d followed by 21 days of depuration) readily accumulated (65)Zn for all three concentrations with an uptake rate constant of 0.160±0.006 L dry weight g(-1) d(-1). Oysters in the dietary assimilation experiment (1h pulse-feed of either (65)Zn-radiolabelled suspended fine-fraction (<63 μm) sediment or the microalgae Tetraselmis sp.) accumulated (65)Zn, with assimilation efficiencies of 59 and 67% for fine sediment and microalgae, respectively. The efflux rates were low for the three experiments (0.1-0.5% d(-1)). A bioaccumulation kinetic model predicts that uptake of Zn will occur predominantly through the dietary ingestion of contaminated fine sediment particles and microalgae within the water column, with considerably greater metal bioaccumulation predicted if oysters ingested microalgae preferentially to sediments. However, the model predicts that for dissolved Zn concentrations greater than 40 μg L(-1), as observed during precipitation events, the uptake of the dissolved phase may contribute ≥50% to accumulation. Overall, the results of the present study suggest that all three sources may be important exposure routes to S. glomerata under different environmental conditions, but contributions from dietary exposure will often dominate.

Acute toxicity testing with the tropical marine copepod Acartia sinjiensis: optimisation and application.

Globally there is limited toxicity data for tropical marine species, and there has been a call for further research and development in the area of tropical marine ecotoxicology. An increase in developmental pressures in northern tropical Australia is causing a higher demand for toxicity test protocols with ecologically relevant species. Copepods are a diverse group of zooplankton that are major components of marine food webs. The calanoid copepod Acartia sinjiensis is widely distributed across tropical and sub-tropical brackish to marine waters of Australia and was identified in a recent comprehensive review of marine tropical toxicity testing in Australia as a suitable test organism. Through a number of optimisation steps including feeding trials, changes to culture and test conditions; a 48-h acute toxicity test with A. sinjiensis was modified to become a highly reliable and reproducible standard test protocol. Control mobility was improved significantly, and the sensitivity of A. sinjiensis to copper (EC50 of 33µg/L), ammonia (EC50 of 10mg/L) and phenol (EC50 of 13mg/L) fell within the ranges of those reported previously, indicating that the modifications did not alter its sensitivity. In a comprehensive literature search we found that this species was the most sensitive to copper out of a range of marine copepods. The test was also successfully applied in toxicity assessments of four environmental samples: two produced formations waters (PFWs) and two mine tailing liquors (MTLs). The toxicity assessments utilised toxicity data from a suite of marine organisms (bacteria, microalgae, copepods, sea urchins, oysters, prawns, and fish). For the PFWs, which were predominantly contaminated with organic chemicals, A. sinjiensis was the most sensitive species (EC50 value 2-17 times lower than for any other test species). For the predominantly metal-contaminated mine tailing liquors, its sensitivity was similar to that of other test species used. The modified 48-h acute toxicity test with A. sinjiensis proved to be a valuable tool in these toxicity assessments, and is recommended for use in tropical marine toxicity assessments for northern Australia.

Geochemical and ecotoxicological assessment of iron‐ and steel‐making slags for potential use in environmental applications

Prior to the productive use of iron- and steel-making slags as environmental amendments, a risk assessment supported by material characterization concomitant with leaching and ecotoxicological testing is necessary. Five iron- and steel-making slags were characterized geochemically, and the leachability of their elemental constituents was assessed. The toxicity of slag leachate to microalgae (Chlorella sp.), cladocerans (Ceriodaphnia dubia), and bacteria (Vibrio fischeri) was related to elemental composition. Slag leachates with the highest concentrations of dissolved elements were the most toxic (10% effective concentration [EC10] ∼1%), whereas those with the lowest concentrations of elements were the least toxic (EC10 63-85%). It was not possible to determine which elements caused the observed toxicity; however, comparisons with contaminant guidelines and published toxicity data identified several elements of potential environmental concern. Low to moderate activities were measured for radionuclides in the U and Th decay chains in slags. Based on these data, some of the slags examined herein are potentially suitable for use as environmental amendments following ≥10 times dilution to ameliorate potential toxic effects because of leachate pH.

Microalgal Toxicity Tests Using Flow Cytometry

These tests determine the inhibitory effects of liquid samples on the growth rate or enzyme activity in freshwater microalgae using flow cytometry as the detector. Flow cytometry is a rapid method for counting and measuring fluorescence and light scattering properties of algal cells at low cell densities, typical of that found in freshwater environments. The test is based on standard phytotoxicity tests (see Chapter 3 of this volume; OECD, 1984; U.S. EPA, 2002) and is applicable to all liquid samples including: wastewaters and effluents, filtered or unfiltered; sediment pore waters; surface waters, groundwaters or leachates; chemicals. The test may be used for screening or definitive tests, alone, or as part of a battery of tests approach. Two tests with the green alga S. capricornutum are described in this chapter: a chronic toxicity test measuring growth rate inhibition using flow cytometry to count algal cells at low cell densities in the presence of particulates;

Copper uptake, intracellular localization, and speciation in marine microalgae measured by synchrotron radiation X-ray fluorescence and absorption microspectroscopy

Metal toxicity to aquatic organisms depends on the speciation of the metal and its binding to the critical receptor site(s) (biotic ligand) of the organism. The intracellular nature of the biotic ligand for Cu in microalgal cells was investigated using the high elemental sensitivity of microprobe synchrotron radiation X-ray fluorescence (SR-XRF) and X-ray absorption near-edge spectroscopy (XANES). The marine microalgae, Ceratoneis closterium, Phaeodactylum tricornutum, and Tetraselmis sp. were selected based on their varying sensitivities to Cu (72-h 50% population growth inhibitions of 8-47 μg Cu/L). Intracellular Cu in control cells was similar for all three species (2.5-3.2 × 10(-15) g Cu/cell) and increased 4-fold in C. closterium and Tetraselmis sp. when exposed to copper, but was unchanged in P. tricornutum (72-h exposure to 19, 40, and 40 μg Cu/L, respectively). Whole cell microprobe SR-XRF identified endogenous Cu in the central compartment (cytoplasm) of control (unexposed) cells. After Cu exposure, Cu was colocated with organelles/granules dense in P, S, Ca, and Si and this was clearly evident in thin sections of Tetraselmis sp. XANES indicated coexistence of Cu(I) and Cu(II) in control and Cu-exposed cells, with the Cu ligand (e.g., phytochelatin) in P. tricornutum different from that in C. closterium and Tetraselmis sp. This study supports the hypothesis that Cu(II) is reduced to Cu(I) and that polyphosphate bodies and phytochelatins play a significant role in the internalization and detoxification of Cu in marine microalgae.

Toxicity of dissolved and precipitated aluminium to marine diatoms

Localised aluminium contamination can lead to high concentrations in coastal waters, which have the potential for adverse effects on aquatic organisms. This research investigated the toxicity of 72-h exposures of aluminium to three marine diatoms (Ceratoneis closterium (formerly Nitzschia closterium), Minutocellus polymorphus and Phaeodactylum tricornutum) by measuring population growth rate inhibition and cell membrane damage (SYTOX Green) as endpoints. Toxicity was correlated to the time-averaged concentrations of different aluminium size-fractions, operationally defined as <0.025μm filtered, <0.45μm filtered (dissolved) and unfiltered (total) present in solution over the 72-h bioassay. The chronic population growth rate inhibition after aluminium exposure varied between diatom species. C. closterium was the most sensitive species (10% inhibition of growth rate (72-h IC10) of 80 (55-100)μg Al/L (95% confidence limits)) while M. polymorphus (540 (460-600)μg Al/L) and P. tricornutum (2100 (2000-2200)μg Al/L) were less sensitive (based on measured total aluminium). Dissolved aluminium was the primary contributor to toxicity in C. closterium, while a combination of dissolved and precipitated aluminium forms contributed to toxicity in M. polymorphus. In contrast, aluminium toxicity to the most tolerant diatom P. tricornutum was due predominantly to precipitated aluminium. Preliminary investigations revealed the sensitivity of C. closterium and M. polymorphus to aluminium was influenced by initial cell density with aluminium toxicity significantly (p<0.05) increasing with initial cell density from 10(3) to 10(5)cells/mL. No effects on plasma membrane permeability were observed for any of the three diatoms suggesting that mechanisms of aluminium toxicity to diatoms do not involve compromising the plasma membrane. These results indicate that marine diatoms have a broad range in sensitivity to aluminium with toxic mechanisms related to both dissolved and precipitated aluminium.