Tests with Daphnia magna: A new approach to prescreen toxicity of newly synthesized acetylcholinesterase reactivators

Reactivators of phosphorylated acetylcholinesterase (oximes) are substances used as a human antidotal therapy for organophosphate poisoning. The objective of our study was to examine if juveniles of freshwater microcrustacean Daphnia magna could be employed as test animals in early screen toxicity tests of those substances as a first step for further experiments with daphnids intoxicated by organophosphates. For this purpose, seven different oximes were investigated. It was found that toxicity of all tested oximes increased with time. Mono-quaternary oximes were approximately ten fold (EC₅₀, 14.9 mg.l^{? 1}) more toxic in 24 hour tests and five fold (EC₅₀ was 79.46 mg.l^{? 1}) more toxic in 48 hour tests than bis-quaternary oximes. Tests with daphnids were shown to be easy to carry out at low cost and provided valuable results which could be used as a starting point for further research.     

Aquatic toxicity test for enchytraeids

It is the purpose of this report to present a simple method for testing the toxicity of chemical substances by using enchytraeids in an aquatic environment. Up to eight different environmental chemicals were applied to various species, mainly of the genus Enchytraeus. The results were compared with those achieved for D. magna. Significant differences, however, between the LC₅₀ values of the various enchytraeid species and the LC₅₀ values for enchytraeids and daphnids could not be observed. For E. cf. buchholzi the toxicological sensitivity of discrete ontogenetic stages was tested. The Aquatic Enchytraeid Test results were compared with those obtained from the Terrestrial Enchytraeid Test. It was found that in soil a chemical could be 600 times less toxic than in water, although the same species (E. albidus) was used in both environments. Even more pronounced were the discrepancies between the terrestrial and aquatic toxicities when the LC₅₀ values for earthworms and daphnids were compared.     

Biological surface coating and molting inhibition as mechanisms of TiO2 nanoparticle toxicity in Daphnia magna

The production and use of nanoparticles (NP) has steadily increased within the last decade; however, knowledge about risks of NP to human health and ecosystems is still scarce. Common knowledge concerning NP effects on freshwater organisms is largely limited to standard short-term (≤48 h) toxicity tests, which lack both NP fate characterization and an understanding of the mechanisms underlying toxicity. Employing slightly longer exposure times (72 to 96 h), we found that suspensions of nanosized (～100 nm initial mean diameter) titanium dioxide (nTiO(2)) led to toxicity in Daphnia magna at nominal concentrations of 3.8 (72-h EC(50)) and 0.73 mg/L (96-h EC(50)). However, nTiO(2) disappeared quickly from the ISO-medium water phase, resulting in toxicity levels as low as 0.24 mg/L (96-h EC(50)) based on measured concentrations. Moreover, we showed that nTiO(2) (～100 nm) is significantly more toxic than non-nanosized TiO(2) (～200 nm) prepared from the same stock suspension. Most importantly, we hypothesized a mechanistic chain of events for nTiO(2) toxicity in D. magna that involves the coating of the organism surface with nTiO(2) combined with a molting disruption. Neonate D. magna (≤6 h) exposed to 2 mg/L nTiO(2) exhibited a "biological surface coating" that disappeared within 36 h, during which the first molting was successfully managed by 100% of the exposed organisms. Continued exposure up to 96 h led to a renewed formation of the surface coating and significantly reduced the molting rate to 10%, resulting in 90% mortality. Because coating of aquatic organisms by manmade NP might be ubiquitous in nature, this form of physical NP toxicity might result in widespread negative impacts on environmental health.     

Evaluation of a high throughput toxicity biosensor and comparison with a Daphnia magna bioassay

A high throughput toxicity biosensor has been designed and constructed using recombinant Escherichia coli cells, containing stress specific promoters (recA, fabA, or katG) or constitutive promoters (lac) fused to luciferase genes originating from Vibrio fisheri. These genetically engineered cells were immobilized in 96 well plates. By optimizing cell immobilization conditions and the strains' response specificity to toxic chemicals, bioluminescent outputs decreased or increased dose-dependently upon adding test chemicals. However, to date the toxicity data obtained using this biosensor have not been compared with the results of other toxicity tests. Phenolics were chosen to evaluate the correlation between the LD50 and the EC50 (GC2) or EC120 (DPD2540) of Daphnia magna and E. coli, respectively. Toxicity data obtained from constitutive strains by bioluminescent level decrements were compared with the results from D. magna as a standard. LD50 values were used as parameters of D. magna toxicity and EC50 of EC120 values were used for the immobilized biosensor. In the DPD2540 test, phenolics, membrane damaging toxic chemicals, for testing immobilized stress specific bacterial strains trigger dose-dependant bioluminescence increase within specific concentration. Although the stress specific responsiveness from the strains could not be compared with D. magna's LD50 values, these responses offer additional information, such as upon the mode of toxic action in the sample, in addition to the cellular toxicity results as indicated by the EC50. This novel high throughput toxicity biosensor can be implemented to investigate the toxicity of any other soluble materials, and can be used as a standardization tool for the evaluation of toxicity.     

邻苯二甲酸酯联合暴露对杜氏盐藻生长的影响

联合毒性效应是指两种或两种以上的化学物质同时或短时期内先后作用于机体所产生的综合毒性作用.论文探讨了典型环境激素邻苯二甲酸二甲酯(DMP)与邻苯二甲酸二乙酯(DEP)对杜氏盐藻(Dunaliella salina )的联合毒性效应.实验以96h的单一毒性效应EC₅₀为一个毒性单位(TU),将上述两种环境激素按照毒性单位比1:1、1:4和4:1的三种比例水平两两组合进行联合毒性试验,研究不同处理下,两种典型环境激素对杜氏盐藻生长的影响.结果表明:两种环境激素对杜氏盐藻生长均有抑制作用,且联合暴露较单一暴露对杜氏盐藻的细胞生长和光合作用抑制更强;DMP和DEP单一暴露对杜氏盐藻的96h EC₅₀分别为317.49mg·L^-1、和69.54mg·L^-1.两者在1:1、1:4和4:1三个比例水平上的联合毒性效应均表现为相加或协同效应,其中比例为1:1的协同效应最强.     

Antifouling activity of simple synthetic isocyanides against larvae of the barnacle Balanus amphitrite

Twelve simple linear isocyanides were synthesized and examined for antifouling activity and toxicity against cyprid larvae of the barnacle, Balanus amphitrite. Larval settlement was inhibited, with EC50 values of 0.046-1.90 microg ml(-1), and they were much less toxic (LD50 values ranging over 21.28 microg ml(-1)) than CuSO4 (EC50 0.30 microg ml(-1) and LD50 2.95 microg ml(-1)). The data indicate that simple linear isocyanides are promising non-toxic antifouling agents.     

Toxicity of landfill leachate to sea urchin development with a focus on ammonia

Sea urchin gametes and embryos serve as a model system to evaluate toxicity in the marine environment. In this study, the toxicity of complex chemical mixtures in leachate samples to sea urchin development was examined with a focus on ammonia, which was the main contaminant of concern in most samples. Two rapid tests, the submitochondrial particle function and bacterial luminescence tests, were also used. Ammonia is highly toxic to sea urchin embryos with an EC50 of 1.3 mg l⁻¹ for the embryos of the Australian sea urchin Heliocidaris tuberculata. Leachate ammonia levels were well above these EC50 concentrations. To assess the contribution of ammonia to leachate toxicity in sea urchin development, we compared the predicted toxic units (PTU) and observed toxic units (OTU) for ammonia for each sample. The PTU/OTU comparison revealed that the sensitivity of the sea urchin embryos to ammonia were altered (enhanced or decreased) by other chemicals in the leachates. This result emphasises the need for parallel chemical analyses and a suite bioassays for evaluating the toxicity of complex and variable chemical mixtures.     

The toxicity of textile reactive azo dyes after hydrolysis and decolourisation

The toxicity of C.I. Reactive Black 5 and three Procion dyes, as found in textile effluents, was determined using the bioluminescent bacterium Vibrio fischeri. Hydrolysed Reactive Black had a slightly greater toxicity than the parent form (EC(50) 11.4+/-3.68 and 27.5+/-4.01 mg l(-1), respectively). A baffled bioreactor with anaerobic and aerobic compartments was used to decolourise hydrolysed Reactive Black 5 in a synthetic effluent. Decolourisation of hydrolysed Reactive Black resulted in an increased toxicity (EC(50) 0.2+/-0.03 mg l(-1)). Toxicity was not detectable when decolourised Reactive Black 5 was metabolised under aerobic conditions. No genotoxicity was detected after the decolourisation of either the parent or the hydrolysed reactive dyes, either in vitro or in the bioreactor. The toxicity and genotoxicity of decolourised C.I. Acid Orange 7 was due to the production of 1-amino-2-naphthol (EC(50) 0.1+/-0.03 mg l(-1)).     

Acclimation of Daphnia magna to environmentally realistic copper concentrations

It may be hypothesised that as the bioavailable background concentration of an essential metal increases (within natural limits), the natural tolerance (to the metal) of the acclimated/adapted organisms and communities will increase. In this study the influence of acclimation to different copper concentrations on the sensitivity of the freshwater cladoceran Daphnia magna Straus was investigated. D. magna was acclimated over three generations to environmentally relevant copper concentrations ranging from 0.5 to 100 microg Cu/l (copper activity: 7.18 x 10(-15) to 3700 x 10(-12) M Cu2+). A modified standard test medium was used as culture and test medium. Medium modifications were: reduced hardness (lowered to 180 mg CaCO3/l) and addition of Aldrich humic acid at a concentration of 5 mg DOC/l (instead of EDTA). The effects of acclimation on these organisms were monitored using acute mortality assays and long-term assays in which life table parameters, copper body concentrations and energy reserves were used as test endpoints. Our results showed a two-fold increase in acute copper tolerance with increasing acclimation concentration for second and third generation organisms. Copper acclimation concentrations up to 35 microg Cu/l (80 pM Cu2+) did not affect the net reproduction and the intrinsic growth rate. The energy reserves of the acclimated daphnids revealed an Optimal Concentration range (OCEE) and concentrations between 5 and 12 microg Cu/l (0.5-4.1 pM Cu2+) and 1 and 35 microg Cu/l (0.023-80 pM Cu2+) seemed to be optimal for first and third generation daphnids, respectively. Lower and higher copper concentrations resulted in deficiency and toxicity responses. It was also demonstrated that up to 35 microg Cu/l, third generation daphnids were able to regulate their total copper body concentration. These results clearly indicate that bioavailable background copper concentrations present in culture media have to be considered in the evaluation of toxicity test results, especially when the toxicity data are used for water quality guideline derivation and/or ecological risk assessment for metals.     

Development of ecotoxicity assay based on inhibition of respiring activity in microbial community using XTT reduction

The aim of this study is to develop ecotoxicity assay for evaluating the influence of chemicals on a microbial ecosystem based on XTT reduction inhibition (XTT assay). XTT reduction method is used for quantification of the microbial respiratory activity. Since the XTT assay indicates the inhibition of microbial respiratory activity, it could evaluate the toxicity of chemicals. Suitable conditions for the XTT assay were determined to be 200 mg/L of particulate organic carbon as test microbe concentration and 15 min of assay time using activated sludge. Toxicities of several chemicals evaluated by activated sludge as test microbes were examined under these conditions. Sensitivity for the toxicity evaluated by the XTT assay using activated sludge microbes was almost the same value was that for the OECD activated sludge respiration inhibition test (ASRI test). XTT assay was also applied for evaluating the influence of chemicals on the soil microbial community and the XTT assay was used to evaluate a median effective concentration (EC(50)) value of 3,5-dichlorophenol (3,5-DCP). The EC(50) value of 3,5-DCP was almost the same as the value using activated sludge as test microbes. These results suggest that the XTT assay using both mixed cultures of non-contaminated environments and chemical extracts from various contaminated environments could evaluate the influence on microbial ecosystems affected by toxic chemicals.     

Toxicity of dissolved Cu, Zn, Ni and Cd to developing embryos of the blue mussel (Mytilus trossolus) and the protective effect of dissolved organic carbon

Marine water quality criteria for metals are largely driven by the extremely sensitive embryo-larval toxicity of Mytilus sp. Here we assess the toxicity of four dissolved metals (Cu, Zn, Ni, Cd) in the mussel Mytilus trossolus, at various salinity levels while also examining the modifying effects of dissolved organic carbon (DOC) on metal toxicity. In 48 h embryo development tests in natural seawater, measured EC50 values were 6.9-9.6 microg L(-1) (95% C.I.=5.5-10.8 microg L(-1)) for Cu, 99 microg L(-1) (86-101) for Zn, 150 microg L(-1) (73-156) for Ni, and 502 microg L(-1) (364-847) for Cd. A salinity threshold of >20 ppt (approximately 60% full strength seawater) was required for normal control development. Salinity in the 60-100% range did not alter Cu toxicity. Experimental addition of dissolved organic carbon (DOC) from three sources reduced Cu toxicity; for example the EC50 of embryos developing in seawater with 20 mg C L(-1) was 39 microg Cu L(-1) (35.2-47.2) a 4-fold increase in Cu EC50. The protective effects of DOC were influenced by their distinct physicochemical properties. Protection appears to be related to higher fulvic acid and lower humic acid content as operationally defined by fluorescence spectroscopy. The fact that DOC from freshwater sources provides protection against Cu toxicity in seawater suggests that extrapolation from freshwater toxicity testing may be possible for saltwater criteria development, including development of a saltwater Biotic Ligand Model for prediction of Cu toxicity.     

The influence of algal densities on the toxicity of chromium for Ceriodaphnia dubia Richard (Cladocera, Crustacea)

Food availability may affect metal toxicity for aquatic organisms. In the present study, the influence of high, medium and low densities of the algae Pseudokirchneriella subcapitata (10(6), 10(5) and 10(4) cells.mL(-1), respectively) on the chronic toxicity of chromium to the cladoceran Ceriodaphnia dubia was investigated. C. dubia was exposed to a range of chromium concentration from 2.71 to 34.04 microg.L(-1) and fed with algae at various densities. In another experiment, the green alga was exposed to chromium concentrations (94 to 774 microg.L(-1)) and supplied as food in different densities to zooplankton. The survival and reproduction of the cladoceran were measured in these toxicity tests. The IC50 for Cr to P. subcapitata and metal accumulated by algal cells were determined. The results of a bifactorial analysis (metal versus algal densities) showed that metal toxicity to zooplankton was dependent on algal densities. Significant toxic effects on the reproduction and survival of C. dubia were observed at 8.73, 18.22 and 34.04 microg.L(-1) Cr when the test organisms were fed with 10(6) cells.mL(-1) of P. subcapitata. Although the chlorophyta retain low chromium content, a decrease in the reproduction and survival of C. dubia occurred when they were fed with high algal density contaminated with 774 microg.L(-1) Cr. It was concluded that high algal density have an appreciable influence on chromium toxicity to daphnids.     

Manganese and zinc toxicity thresholds for mountain and Geyer willow

Information on the heavy metal toxicity thresholds of woody species endemic to the western United States is lacking but critical for successful restoration of contaminated riparian areas. Manganese (Mn, 50-10,000 mg l(-1)) and zinc (Zn, 100-1000 mg l(-1)) toxicity thresholds were determined for Geyer (Salix geyeriana Anderss.) and mountain (S. monticola Bebb) willow using a sand-culture technique. The lethal concentration (50%) values were 3117 and 2791 mg Mn l(-1) and 556 and 623 mg Zn l(-1) for Geyer and mountain willow, respectively. The effective concentration (50%) values for shoots were 2263 and 1027 mg Mn l(-1) and 436 and 356 mg Zn l(-1) for Geyer and mountain willow, respectively. Shoot tissue values did not increase with increasing treatment concentrations. However, metals in the roots did increase consistently in response to the treatments. Metal levels in the shoot tissues were low for Zn (65-139 mg kg(-1)) and moderate for Mn (1300-2700 mg kg(-1)). Geyer and mountain willow have good resistance to Mn, possibly due to evolution in hydric soils with increased Mn availability, and may be useful for phytostabilization of soils with high levels of available Mn. Both species were affected to a greater degree by Zn as compared to Mn, but still exhibited good resistance and should be useful in remediating sites with at least moderate levels of available Zn. Based on the thresholds evaluated, Geyer willow had greater resistance to both Mn and Zn as compared to mountain willow, especially at lower concentrations in which growth of Geyer willow was actually stimulated.     

Biological treatment of 2,4-dichlorophenol containing synthetic wastewater using a rotating brush biofilm reactor

A newly developed rotating brush biofilm reactor was used for DCP, COD and toxicity removal from 2,4-dichlorophenol (DCP) containing synthetic wastewater at different feed COD, TCP concentrations and A/Q (biofilm surface area/feed flow rate) ratios. A Box-Wilson statistical experiment design was used by considering the feed DCP (50-500 mg l(-1)), COD (2000-6000 mg l(-1)) and A/Q ratio (73-293 m2 d m(-3)) as the independent variables while percent DCP, COD, and toxicity removals were the objective functions. The experimental data were correlated by a quadratic response function and the coefficients were determined by regression analysis. Percent DCP, COD and toxicity removals calculated from the response functions were in good agreement with the experimental data. DCP, COD and toxicity removals increased with increasing A/Q ratio and decreasing feed DCP concentrations. The optimum A/Q ratio resulting in the highest COD (90%), DCP (100%) and toxicity (100%) removals with the highest feed COD (6000 mg l(-1)) and DCP (500 mg l(-1)) contents was nearly 210 m2 d m(-3).     

Toxic Bacillus pumilus from indoor air, recycled paper pulp, Norway spruce, food poisoning outbreaks and clinical samples

Forty-four B. pumilus isolates of food poisoning, clinical, environmental and industrial origins were investigated for toxin production using the boar spermatozoan motility assay, previously shown to be a sensitive method for detecting non-protein toxins from B. cereus and B. licheniformis. The three toxic isolates originated from live tree, indoor air and recycled paper pulp and were more toxic than the previously described food poisoning isolates of B. licheniformis, whereas the B. pumilus food poisoning and clinical isolates were lower in toxicity. The type strain also produced inhibitory substances. The toxic substances were insensitive to heat (100 degrees C, 20 min), to pH 2 or pH 10 and to digestion with pronase. The substances were readily soluble in methanol and chloroform, but less soluble in toluene. Exposure of boar spermatozoa to 1-10 microg ml(-1) (EC50) of methanol soluble substance from the four strains disrupted the plasma membrane permeability barrier, induced abnormalities in the postacrosomal sheath, collapsed the mitochondrial and suppressed cytoplasmic NAD reduction. No change was observed in human peripheral blood lymphocytes exposed to concentrations of B. pumilus extract that affected spermatozoa. The toxin producing isolates were 99.4 to 99.6% similar in 16SrDNA (500 bp) to the type strain and could not be distinguished from the 41 non-toxic isolates by biochemical properties or whole cell fatty acid composition.     

Physiological effects of chronic silver exposure in Daphnia magna

Daphnia magna were exposed to a total concentration of 5.0+/-0.04 microg Ag/l, added as AgNO(3) (dissolved concentration, as defined by 0.45 microm filtration = 2.0+/-0.01 microg Ag/l) in moderately hard synthetic water under static conditions (total organic carbon = 4.80+/-1.32 mg/l) with daily feeding and water renewal, for 21 days. There was no mortality in control daphnids and 20% mortality in silver-exposed animals. Silver exposure caused a small but significant reduction of reproductive performance manifested as a 13.7% decrease in the number of neonates produced per adult per reproduction day over the 21-day exposure. However, silver exposed daphnids also exhibited a much more marked ionoregulatory disturbance, which was characterized by a 65% decrease in whole body Na(+) concentration, and an 81% inhibition of unidirectional whole body Na(+) uptake. Previous work on the acute toxicity of Ag(+) to daphnids has shown that Na(+) uptake inhibition is directly related to inhibition of Na(+),K(+)-ATPase activity. Therefore, we suggest that the Na(+) uptake inhibition seen in response to chronic silver exposure was explained by an inhibition of the Na(+) channels at the apical 'gill' membrane, since a 60% increase in whole body Na(+),K(+)-ATPase activity was observed in the chronically silver-exposed daphnids. Our findings demonstrate that, in broad view, the key mechanism involved in chronic silver toxicity in D. magna, the most acutely sensitive freshwater organism tested up to now, resembles that described for acute toxicity-i.e. ionoregulatory disturbance associated with inhibition of active Na(+) uptake, though the fine details may differ. Our results provide encouragement for future extension of the current acute version of the Biotic Ligand Model (BLM) to one that predicts chronic silver toxicity for environmental regulation and risk assessment. The results strongly suggest that Na(+) uptake inhibition is the best endpoint to determine sensitivity to both acute and chronic toxicity in the scope of future versions of the BLM for silver.     

Oral toxicity of p-aminopropiophenone to brushtail possums (Trichosurus vulpecula), dama wallabies (Macropus eugenii), and mallards (Anas platyrhynchos)

Development of p-aminopropiophenone (PAPP) as a toxicant for pest predator management in New Zealand and Australia prompted investigation of its toxicity to potential nontarget species. Acute oral toxicity of PAPP in brushtail possums (Trichosurus vulpecula), dama wallabies (Macropus eugenii), and Mallards (Anas platyrhynchos) was estimated in pen trials, carried out between February 2000 and September 2001. The susceptibility of possums (LD50>or=500 mg kg(-1)) and wallabies (LD50 89 mg kg(-1)) to PAPP was low in comparison to noncarnivorous placental mammal species, but ducks (LD50 38 mg kg(-1)) were more susceptible than other bird species. These results suggest that the nontarget hazard to possums and wallabies from PAPP bait applied for pest predator control would be low. However, future development of PAPP as a vertebrate pest control agent should include rigorous assessments of the hazard posed by bait formulations to bird species and provision for delivery techniques that could mitigate exposure of nontarget birds.     

叔丁基对羟基茴香醚和诺氟沙星对水生生物的影响

采用急性毒性试验的方法,研究了叔丁基对羟基茴香醚(Butylated hydroxyanisole,BHA)和诺氟沙星(Norfloxacin,NFLX)对水生生物斜生栅藻和大型溞的毒性效应。结果表明大型溞在BHA和NFLX暴露下48h的LC₅₀分别为3.15mg·L^-1和194.98mg·L^-1。BHA和NFLX对斜生栅藻也有明显的毒性作用,其96h的EC₅₀分别为6.19mg·L^-1和50.18mg·L^-1。大型蚤对BHA暴露的敏感性强于斜生栅藻,而斜生栅藻对NFLX的敏感性比大型溞强。根据化学物质对鱼类和溞类的毒性评价标准,BHA和NFLX分别属于中等和低等毒性的化合物。     

浮选剂丁基黄原酸钠对草鱼早期发育阶段的毒性效应

丁基黄原酸钠是一种金属矿的浮选剂.作者研究了它对草鱼早期发育阶段(孵化、存活、生长、畸形等)的毒性效应.结果表明,丁基黄原酸钠对草鱼胚胎96h的半致死浓度为17.8mg/L,致畸半效应浓度为8.85mg/L,当浓度为24mg/L时,其胚胎的孵化率仅为38.8%,对生长有影响的浓度为5.6-10.0mg/L,浓度在5.6mg/L就可诱发畸形.丁基黄原酸钠对草鱼胚胎具有强烈的致崎作用,崎形的主要症状为弯体和体表瘤状赘生物.