Development and application of whole-sediment toxicity test using immobilized freshwater microalgae Pseudokirchneriella subcapitata

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 CaCl(2) 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.     

Development and field validation of a biotic ligand model predicting chronic copper toxicity to Daphnia magna

In this study, we developed a toxicity model predicting the long-term effects of copper on the reproduction of the cladoceran Daphnia magna that is based on previously reported toxicity tests in 35 exposure media with different water chemistries. First, it was demonstrated that the acute copper biotic ligand model (BLM) for D. magna could not serve as a reliable basis for predicting chronic copper toxicity. Consequently, BLM constants for chronic exposures were derived by multiple regression analysis of 21-d median effective concentrations (EC50s; expressed as Cu2+ activity) versus physicochemistry from a large toxicity dataset and the results of an additional experiment in which the individual effect of sodium on copper toxicity was investigated. The effect of sodium on chronic toxicity (log K NaBL = 2.91) seemed to be similar to its effect on acute toxicity (log K NaBL = 3.19). However, in contrast to the acute BLM, no significant calcium, magnesium, or combined competition effect was observed, and an increase in proton competition and bioavailability of CuOH+ and CuCO3 complexes was noted. Some indirect evidence was also found for some limited toxicity of complexes of copper with two of three tested types of dissolved organic matter. Because the latter was only a minor effect, this factor was not included in the chronic Cu BLM. The newly developed model performed well in predicting 21-d EC50s and no-observed-effect concentrations in natural water samples: 79% of the toxicity threshold values were predicted within a factor of two of the observed values. It is clear, however, that more research is needed to provide information on the exact mechanisms that have resulted in different BLM constants for chronic exposures (as opposed to acute exposures). It is suggested that the developed model can contribute to the improvement of risk assessment procedures of copper by incorporating bioavailability of copper in these regulatory exercises.     

Toxicity assessment of 40 herbicides to the green alga Raphidocelis subcapitata

The effects of 40 herbicides with nine modes of action on the green alga Raphidocelis subcapitata were studied by 96-h acute toxicity tests. Results showed that the EC50 of the herbicides with respect to the photosynthetic processes of R. subcapitata ranged from 0.0007 to 4.2286 mgL(-1). Photosynthesis was the process of the green alga most sensitive to the tested herbicides. The most toxic herbicides were atrazine, ametryme, simazine, prometryne, cyanazine, isoproturon, chlorotoluron, diuron, methabenzthiazuron, and paraquat. The EC50 of the protoporphyrinogen oxidase inhibitor oxadiargyl, which was the parameter least sensitive to the herbicides tested in this study, was 42.5 mgL(-1). The descending order of the average acute toxicity to R. subcapitata of herbicides with regard to the nine modes of action was as follows: photosynthetic process>cell division>lipid synthesis, acetyl-coenzyme A carboxylase>acetolactate synthase> 5-enolpyruvyl-shikimate-3-phosphate synthase, glutamine synthase, hormone synthesis>protoporphyrinogen oxidase.     

Influence of chloride on silver uptake by two green algae, Pseudokirchneriella subcapitata and Chlorella pyrenoidosa

Silver bioavailability in the presence of chloride was estimated from short-term (< or = 60 min) uptake experiments with two green algae, Pseudokirchneriella subcapitata and Chlorella pyrenoidosa. In the first experiment, silver uptake was monitored under two concentration regimes in which total dissolved silver ([Ag]tot) and [Cl] were manipulated to maintain [Ag+] at a constant value (10 nM). Comparable uptake rates were measured for both treatments despite the dramatic changes in [Cl] and [Ag]tot. In the second experiment, ambient [Ag]tot was held constant (10 or 115 nM), but [Cl] was varied (0.005-50 mM) to explore the whole range of silver chloro-complexes. Intracellular silver varied markedly along the [Cl] gradient and exhibited a clear, positive correlation with ambient [Ag+] for both algae. We conclude that the biotic ligand model reliably describes silver bioavailability in the presence of chloride for the two test algae and that its applicability depends on the relative magnitudes of silver fluxes through the unstirred diffusion layer and across the cell membrane, with the latter being affected by the presence or absence of a Cu(I) transporter. In the presence of chloride, no evidence was found for the internalization of silver via anion transport or passive diffusion of the neutral mono-chloro-complex, AgCl0.     

Effects of natural water chemistry on nanosilver behavior and toxicity to Ceriodaphnia dubia and Pseudokirchneriella subcapitata

The success of nanotechnology will undoubtedly lead to the increased introduction of nanoparticles (NPs) into natural systems, and aquatic systems will likely act as sinks for these new pollutants. Differences in the chemistry of these aquatic systems will control changes in surface properties of NPs and therefore could impact their environmental fate and toxicity. A series of batch experiments was conducted to determine the effect of natural waters with different solution chemistries on nanosilver (nAg) particle dispersion, stability, and toxicity. Toxicity assays were performed in tested natural waters as well as in traditional growth media. Briefly, nAg suspended in a natural water with a low ionic strength/dissolved organic carbon (DOC) ratio had an average particle size of 76.8?±?0.4?nm and was less toxic to Ceriodaphnia dubia and Pseudokirchneriella subcapitata (median lethal concentration [LC50]?=?221?ppb and median inhibition concentration [IC50]?=?1,600?ppb, respectively), whereas a natural water with a high ionic strength/DOC ratio had an average particle size of 192?±?5.1?nm and produced a much higher toxicity response (LC50?=?0.433?ppb and IC50?=?22.6?ppb, respectively). These findings help to establish needed correlations between water-matrix-dependent nAg particle properties and toxicity implications and indicate that the use of traditional growth media in toxicity assays concerning engineered NPs might not always be appropriate.     

Acute toxicity of ionic liquids for three freshwater organisms: Pseudokirchneriella subcapitata, Daphnia magna and Danio rerio

The static acute toxicities of 18 ionic liquids (ILs) were determined for three representative freshwater organisms, the cladoceran Daphnia magna, the green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), and the fish Danio rerio (formerly known as zebrafish). The test kit compounds contained three widely used ILs (1-butyl-3-methylimidazolium bis(triflimide), [bmim][Tf₂N], butylpyridinium bis(triflimide), [bpy][Tf₂N], and N,N-methylbutylpyrrolidinium bis(triflimide), [bmpyrr][Tf₂N]) and 15 less common salts. These latter comprised a range of five anions, four positively charged head groups (ammonium, morpholinium, thiophenium, and sulfonium), five 1-methyl-3-alkyl imidazolium derivatives bearing a specific functional group on the longer alkyl chain (Cl, OH, or (CH₃)₃Si) and three imidazolium derivatives characterized by the presence of a hydrogen atom on the imidazolium nitrogen (“Brønsted acidic imidazolium”-based ILs). Generally, long-chain ammonium salts showed higher toxicity to algae, cladocerans, and fish, whereas very low toxicities characterized sulfonium- and morpholinium-based ILs. In imidazolium-based ILs, the substitution of one or two carbon atoms of the longer alkyl chain with a more electronegative atom (chlorine or oxygen) reduced the acute toxicity for algae and cladocerans. Low toxicity also characterized the “Brønsted acidic imidazolium”-based ILs. Structural information for a rational designer of safer ILs can be obtained from these studies.     

An in situ bioassay for freshwater environments with the microalga Pseudokirchneriella subcapitata

This study was designed to evaluate the suitability of an in situ microalgal bioassay with Pseudokirchneriella subcapitata to be used in freshwater environments. The assay potential was investigated by deploying it in a system impacted by acid mine drainage. Water samples were collected to perform a laboratory assay also. P. subcapitata was viewed to be a good option for the in situ assay because it grew well and according to control acceptability criteria when immobilized in calcium alginate beads. A reduction in algal growth was apparent at both impacted sites demonstrating assay sensitivity: the site closest to the effluent discharge was clearly impacted and the one further downstream appeared to be moderately impacted. Results from the laboratory assay, designed to distinguish effects of nutrient differences across sites from those due to the effluent, confirmed the in situ responses. Results are discussed in light of the significance of combining information from different assessment tools, namely in situ and laboratory assays, as well as water-quality parameters, particularly at sites that are moderately impacted.     

Toxicity and quantitative structure-activity relationships of nitriles based on Pseudokirchneriella subcapitata

This study presents the toxicity data of various nitriles to Pseudokirchneriella subcapitata using a closed algal toxicity testing technique with no headspace. Two different response endpoints, i.e., dissolved oxygen (DO) production and algal growth rate, were used to evaluate the toxicity of nitriles. In general, the DO endpoint revealed higher inhibitory effects than that from algal growth rate. Furthermore, halogen-substituted nitriles were found to be extremely toxic to P. subcapitata. With increasing numbers of the halogen atoms, stronger toxicity was observed. The bromine substitutent also seems to be more toxic than chlorine substitutent. Quantitative structure-activity relationships (QSARs) were established based on the chemicals' Elumo values and hydrophobicity (logK(ow)). Such relationships may thus be useful in predicting the toxicity of other compounds of the same mode of toxic action. Furthermore, for various aquatic organisms, the relative sensitivity relationship is: Pimephales promelas > or = P. subcapitata> Tetrahymena Pyriformis>Daphnia magna>luminescent bacteria (Microtox). The alga, P. subcapitata, was found to be quite sensitive to nitriles compared to other organisms.     

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.     

Combination of a higher-tier flow-through system and population modeling to assess the effects of time-variable exposure of isoproturon on the green algae Desmodesmus subspicatus and Pseudokirchneriella subcapitata

A flow-through system was developed to investigate the effects of time-variable exposure of pesticides on algae. A recently developed algae population model was used for simulations supported and verified by laboratory experiments. Flow-through studies with Desmodesmus subspicatus and Pseudokirchneriella subcapitata under time-variable exposure to isoproturon were performed, in which the exposure patterns were based on the results of FOrum for Co-ordination of pesticide fate models and their USe (FOCUS) model calculations for typical exposure situations via runoff or drain flow. Different types of pulsed exposure events were realized, including a whole range of repeated pulsed and steep peaks as well as periods of constant exposure. Both species recovered quickly in terms of growth from short-term exposure and according to substance dissipation from the system. Even at a peak 10 times the maximum predicted environmental concentration of isoproturon, only transient effects occurred on algae populations. No modified sensitivity or reduced growth was observed after repeated exposure. Model predictions of algal growth in the flow-through tests agreed well with the experimental data. The experimental boundary conditions and the physiological properties of the algae were used as the only model input. No calibration or parameter fitting was necessary. The combination of the flow-through experiments with the algae population model was revealed to be a powerful tool for the assessment of pulsed exposure on algae. It allowed investigating the growth reduction and recovery potential of algae after complex exposure, which is not possible with standard laboratory experiments alone. The results of the combined approach confirm the beneficial use of population models as supporting tools in higher-tier risk assessments of pesticides.     

Effects of Nutrients and Salinity on the Algal Assay Using Pseudokirchneriella subcapitata (Korshikov) Hindak

Bulletin of Environmental Contamination and Toxicology -     

Photo-induced toxicity of anthracene to the green alga,Selenastrum capricornutum

The photo-induced toxicity of the polycyclic aromatic hydrocarbon (PAH), anthracene, to the green alga, Selenastrum capricornutum, was characterized. The dose-response relationships among anthracene concentration, ultraviolet (UV) radiation intensity, and algal growth rate and ¹⁴C-bicarbonate incorporation were determined. The 22 h EC₅₀ for specific growth rate was inversely related to UV-A radiation intensity and ranged from 37.4 to 3.9 g/L anthracene. For ¹⁴C-bicarbonate incorporation on a volume basis (primary production), the 24 h EC₅₀ ranged from 24.0 to 3.3 g/L anthracene depending on the UV-A intensity. The incorporation of ¹⁴C-bicarbonate on a per cell basis (cellular photosynthesis) was more resistant than cell growth or primary production. The threshold for photo-induced toxicity of anthracene was 1.5–3 g/L anthracene, however, no UV-A radiation threshold was evident for many of the measurements of toxicity studied. Algae appeared to be slightly more resistant to photo-induced toxicity of anthracene than fishes and invertebrates. An environmental hazard assessment suggests that some aquatic systems are sufficiently contaminated by PAH that a hazard to natural algal communities due to photo-induced toxicity of PAH may be present.     

Toxicity of fluorotelomer carboxylic acids to the algae Pseudokirchneriella subcapitata and Chlorella vulgaris, and the amphipod Hyalella azteca

Perfluorinated acids (PFAs) have elicited significant global regulatory and scientific concern due to their persistence and global pervasiveness. A source of PFAs in the environment is through degradation of fluorotelomer carboxylic acids (FTCAs) but little is known about the toxicity of these degradation products. Previous work found that FTCAs were two to three orders of magnitude more toxic to some freshwater invertebrates than their PFA counterparts and exhibited comparable chain-length-toxicity relationships. In this study, we investigated the toxicity of the 6:2, 8:2, and 10:2 saturated (FTsCA) and unsaturated (FTuCA) fluorotelomer carboxylic acids to two species of freshwater algae, Chlorella vulgaris and Pseudokirchneriella subcapitata, and the amphipod, Hyalella azteca. C. vulgaris was generally the most sensitive species, with EC₅₀s of 26.2, 31.8, 11.1, and 4.2 mg/L for the 6:2 FTsCA, 6:2 FTuCA, 8:2 FTuCA, and 10:2 FTsCA, respectively. H. azteca was most sensitive to the 8:2 FTsCA and 10:2 FTuCA, with LC₅₀s of 5.1 and 3.7 mg/L. The toxicity of the FTCAs generally increased with increasing carbon chain length, and with saturation for most of the species tested, with the exception of P. subcapitata, which did not exhibit any trend. These observations agree with chain-length-toxicity relationships previously reported for the PFCAs and support the greater toxicity of the FTCAs compared to PFCAs. However, the toxicity values are approximately 1000-fold above those detected in the environment indicating negligible risk to aquatic invertebrates.     

Effects of sulfate on selenate uptake and toxicity in the green alga Selenastrum capricornutum

Sulfate and selenate compete for active transport across cell membranes via a common permease, suggesting that a significant interaction may exist between uptake of the two ions. The effect of sulfate on selenate uptake and toxicity in Selenastrum capricornutum was investigated using two sulfate levels (3.3 and 33 mg/L S, as SO₄) and two selenate levels (10 and 100 g/L Se, as SeO₄). This provided four treatments: one with S:Se molar ratio of 75, two with molar ratios of 750, and one with a molar ratio of 7,500. Selenium uptake and toxicity analyses demonstrated antagonism between the two anions. Increasing sulfate resulted in significantly reduced selenate uptake and increased algal growth. There was a significant difference in selenate uptake between the two treatments with the same S:Se molar ratio suggesting different relative permease affinities for each of the ions at different substrate levels (i.e., above and below permease saturation levels) and/or the presence of two different permease systems. The environmental significance of sulfate and selenate antagonism is discussed.     

Antioxidative role of nitric oxide on copper toxicity to a chlorophycean alga, Chlorella

The response of Chlorella vulgaris to copper exposure was investigated under laboratory batch culture conditions. Increased toxicity of Cu with respect to photosynthetic carbon fixation, O(2) evolution, chlorophyll fluorescence, and oxidative burst was observed for N-NH(4)(+)-grown cultures. The addition of sodium nitroprusside, a nitric oxide (NO) donor, in combination with Cu to N-NH(4)(+)-grown Chlorella not only lowered the inhibition levels of carbon fixation, O(2) evolution, and maximum quantum yield of PS II, but also significantly reduced the oxidative burst. The protective action of sodium nitroprusside was, however, arrested in cultures in which sodium nitroprusside was supplemented in combination with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a specific scavenger of NO in the experimental system. The N-NO(3)(-)-grown Chlorella depicted less sensitivity to Cu compared to its N-NH(4)(+)-grown counterpart. The N-NO(3)(-)-, N-NH(4)(+)-, and N-NH(4)(+)+sodium nitroprusside-grown Chlorella did not show any significant differences with respect to their Cu uptake potential. The role of NO as an antioxidant is discussed.     

Critical evaluation and further development of methods for testing ecotoxicity at multiple pH using Daphnia magna and Pseudokirchneriella subcapitata

To meet the requirements of risk assessment legislature regarding the ecotoxicity of ionizing compounds, the present study attempts to establish easy, robust methods for testing ecotoxicity at various pH levels. An overview is given of the buffering methods found in the literature. This is supplemented by a series of experiments where toxicity and ability to stabilize pH of seven common buffering compounds was tested on Daphnia magna and Pseudokirchneriella subcapitata. We consider a buffer applicable at a given concentration if the pH drift is below 0.2 pH units, and if there are no toxic effects. Twenty-four- and 48-h acute toxicity tests with D. magna were carried on a series of organic buffers with pH monitoring. Based on the experimental results it is possible to give recommendations for buffer concentrations for use in toxicity testing with D. magna at pH levels in the range of pH 6.0-7.8 for 48 h exposure, and pH 6.0-9.5 for 24 h exposure. Forty-eight- and 72-h growth inhibition tests with P. subcapitata were carried out, and recommendations for buffer concentrations at pH 7.5 and 8.0 are made for both 48 and 72 h of exposure.     

Growth Stimulation and Inhibition Effects of 4-Hydroxybenzoic Acid and Some Related Compounds on the Freshwater Green Alga Pseudokirchneriella subcapitata

4-Hydroxybenzoic acid (4-HBA) exhibited low algal toxicity with the 72-h median inhibition concentration (IC₅₀) of 9.9 mmol/L in the standard growth inhibition test using the freshwater green alga Pseudokirchneriella subcapitata. In contrast, it stimulated the algal growth at lower concentrations ranging from 0.1 to 1.0 mmol/L. Comparative studies with benzoic acid and 2- and 3-hydroxybenzoic acids (2-HBA and 3-HBA) indicated that 2-HBA was the most toxic, giving a 72-h IC₅₀ of 0.172 mmol/L, and 4-HBA was the least toxic and that only 4-HBA had the pronounced growth stimulation activity. In a semicontinuous exposure to 4-HBA (0.15 and 0.3 mmol/L), algae maintained increased cell growth compared with controls during up to 10 times consecutive batch cultures, without any indication of adaptive responses to the growth enhancing effect of 4-HBA. Return to the clean standard medium of the exposed cells resulted in the quick recovery from the stimulant effect. Furthermore, 4-HBA (0.3 mmol/L) was found to diminish the toxicity of 2-HBA (< 1.0 mmol/L). The presence of such compounds in the complex mixture might interact with other toxicants to alter the whole toxicity when assessed by the algal growth inhibition test. The effects of 4-HBA on P. subcapitata growth observed in the present study are not expected for planktonic algae in the aquatic environments, because known environmental concentrations are far below the effective concentration range.