A fluorescence-based bioassay for aquatic macrophytes and its suitability for effect analysis of non-photosystem II inhibitors

Background, Goals and Scope During the last years the miniaturization of toxicity test systems for rapid and parallel measurements of large quantities of samples has often been discussed. For unicellular algae as well as for aquatic macrophytes, fluorescence-based miniaturized test systems have been introduced to analyze photosystem II (PSII) inhibitors. Nevertheless, high-throughput screening should also guarantee the effect detection of a broad range of toxicants in order to ensure routinely applicable, high-throughput measuring device experiments which can cover a broad range of toxicants and modes of action others than PSII inhibition. Thus, the aim of this study was to establish a fast and reproducible measuring system for non-PSII inhibitors for aquatic macrophyte species to overcome major limitations for use. Methods A newly developed imaging pulse-amplitude-modulated chlorophyll fluorometer (I-PAM) was applied as an effect detector in short-term bioassays with the aquatic macrophyte species Lemna minor. This multiwell-plate based measuring device enabled the incubation and measurement of up to 24 samples in parallel. The chemicals paraquat-dichloride, alizarine and triclosan were chosen as representatives for the toxicant groups of non-PSII herbicides, polycyclic aromatic hydrocarbons (PAHs) and pharmaceuticals and personal care products (PPCPs), which are often detected in the aquatic environment. The I-PAM was used (i) to establish and validate the sensitivity of the test system to the three non-PSII inhibitors, (ii) to compare the test systems with standardized and established biotests for aquatic macrophytes, and (iii) to define necessary time scales in aquatic macrophyte testing. For validation of the fluorescence-based assay, the standard growth test with L. minor (ISO/DIS 20079) was performed in parallel for each chemical. Results The results revealed that fluorescence-based measurements with the I-PAM allow rapid and parallel analysis of large amounts of aquatic macrophyte samples. The I-PAM enabled the recording of concentration-effect-curves with L. minor samples on a 24-well plate with single measurements. Fluorescence-based concentration-effect-curves could be detected for all three chemicals after only 1 h of incubation. After 4–5 h incubation time, the maximum inhibition of fluorescence showed an 80–100% effect for the chemicals tested. The EC50 after 24 h incubation were estimated to be 0.06 mg/L, 0.84 mg/L and 1.69 mg/L for paraquatdichloride, alizarine and triclosan, respectively. Discussion The results obtained with the I-PAM after 24 h for the herbicide paraquat-dichloride and the polycyclic aromatic hydrocarbon alizarine were in good accordance with median effective concentrations (EC50s) obtained by the standardized growth test for L. minor after 7 d incubation (0.09 mg/L and 0.79 mg/L for paraquat-dichloride and alizarine, respectively). Those results were in accordance with literature findings for the two chemicals. In contrast, fluorescence-based EC50 of the antimicrobial agent triclosan proved to be two orders of magnitude greater when compared to the standard growth test with 7 d incubation time (0.026 mg/L) as well as with literature findings. Conclusion Typically, aquatic macrophyte testing is very time consuming and relies on laborious experimental set-ups. The I-PAM measuring device enabled fast effect screening for the three chemicals tested. While established test systems for aquatic macrophytes need incubation times of ≥ 7 d, the I-PAM can detect inhibitory effects much earlier (24 h), even if inhibition of chemicals is not specifically associated with PSII. Thus, the fluorescence-based bioassay with the I-PAM offers a promising approach for the miniaturization and high-throughput testing of chemicals with aquatic macrophytes. For the chemical triclosan, however, the short-term effect prediction with the I-PAM has been shown to be less sensitive than with long-term bioassays, which might be due to physicochemical substance properties such as lipophilicity. Recommendations and Perspectives The results of this study show that the I-PAM represents a promising tool for decreasing the incubation times of aquatic macrophyte toxicity testing to about 24 h as a supplement to existing test batteries. The applicability of this I-PAM bioassay on emergent and submerged aquatic macrophyte species should be investigated in further studies. Regarding considerations that physicochemical properties of the tested substances might play an important role in microplate bioassays, the I-PAM bioassay should either be accompanied by evaluating physicochemical properties modeled from structural information prior to an experimental investigation, or by intensified chemical analyses to identify and determine nominal concentrations of the toxicants tested. The chemicals paraquat-dichloride, alizarine and triclosan were chosen as representatives for the toxicant groups of non-PSII herbicides, PAHs and PPCPs which are often detected in the aquatic environment. Nevertheless, in order to ensure a routinely applicable measuring device, experiments with a broader range of toxicants and samples of surface and/or waste waters are necessary. ESS-Submission Editor: Dr. Markus Hecker (MHecker@Entrix.com)     

土壤中4-溴苯胺降解动力学及其生态毒性追踪

秀谷隆是一种被广泛应用的取代脲类除草剂，其主要降解产物为４－溴苯胺（４－ＢＡ），研究４－溴苯胺在不同土壤中的降解动力学规律，并用土壤环境指示动物弹尾目跳虫Ｃｏｌｌｅｍｂｏｌａ（Ｆｏｌｓｏｍｉａ ｃａｎｄｉｄａ）跟踪指示该化合物在进一步降解过程中的毒性，结果表明：４－溴苯胺在土壤中的降解行为可用一级反应动力学方程来较好地描述，其生态毒性明显高于母体化合物秀谷隆，该化合物在粘土中的残留量低于砂土，但毒     

Changing effluent chemistry affect survival,growth and physiological function of <Emphasis Type="Italic">Acacia nilotica</Emphasis> seedlings in northwestern region of India

Recycling and conservation efforts for water are the need of the day because of the lack of new water sources and the ever-increasing demand for drinking water. Seedlings of Acacia nilotica L. were irrigated with: canal water (T₁, control); municipal effluent (T₂); textile effluent (T₃); steel effluent (T₄); textile + municipal effluent in 1:1 ratio (T₅); steel + municipal effluent in 1:2 ratio (T₆); steel + textile in 1:2 ratio (T₈) and steel + municipal + textile in 1:2:2 ratio (T₇) with views to observe effluents effect on the seedlings and its adaptability and to recommend safe disposal of these effluents. Seedlings in T₆, T₇ and T₈ showed 50% lesser height and collar diameter than those in control. Seedlings in T₂ attained greatest height, collar diameter, numbers of branches and produced 140 g dry biomass seedling⁻¹. Highest concentration of manganese (Mn), iron (Fe), copper (Cu) and zinc (Zn) and lowest concentration of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca) and magnesium (Mg) in the seedlings of T₄, T₆, T₇ and T₈ resulted in nutritional imbalance, mineral toxicity and reduction in photosynthetic (Pn) and transpiration (E) rates and caused seedling mortality. Seedlings of T₃ had highest sodium concentration and low concentration of Ca, Mg and micronutrients resulting in nutritional imbalance, augmented chlorosis and reduced gas exchange and biomass by half as compared to control. Increased growth, Pn and E and biomass in seedlings of T₅ over T₃ and survival period in T₆, T₇ and T₈ seedlings suggested a beneficial effect of effluents mixing. Unscientific disposal should be avoided and toxic concentration of metal ions␣may be reduced for long-term application and harmless disposal of effluents in afforestation and urban development.     

Cd-tolerant Suillus luteus: A fungal insurance for pines exposed to Cd

Soil metal pollution can trigger evolutionary adaptation in soil-borne organisms. An in vitro screening test showed cadmium adaptation in populations of Suillus luteus (L.: Fr.) Roussel, an ectomycorrhizal fungus of pine trees. Cadmium stress was subsequently investigated in Scots pine (Pinus sylvestris L.) seedlings inoculated with a Cd-tolerant S. luteus, isolated from a heavy metal contaminated site, and compared to plants inoculated with a Cd-sensitive isolate from a non-polluted area. A dose-response experiment with mycorrhizal pines showed better plant protection by a Cd-adapted fungus: more fungal biomass and a higher nutrient uptake at high Cd exposure. In addition, less Cd was transferred to aboveground plant parts. Because of the key role of the ectomycorrhizal symbiosis for tree fitness, the evolution of Cd tolerance in an ectomycorrhizal partner such as S. luteus can be of major importance for the establishment of pine forests on Cd-contaminated soils.     

Polychlorinated naphthalenes in human adipose tissue from New York, USA

Polychlorinated naphthalenes (PCNs) are persistent, bioaccumulative, and toxic contaminants. Prior to this study, the occurrence of PCNs in human adipose tissues from the USA has not been analyzed. Here, we have measured concentrations of PCNs in human adipose tissue samples collected in New York City during 2003-2005. Concentrations of PCNs were in the range of 61-2500 pg/g lipid wt. in males and 21-910 pg/g lipid wt. in females. PCN congeners 52/60 (1,2,3,5,7/1,2,4,6,7) and 66/67 (1,2,3,4,6,7/1,2,3,5,6,7) were predominant, collectively accounting for 66% of the total PCN concentrations. Concentrations of PCNs in human adipose tissues were 2-3 orders of magnitude lower than the previously reported concentrations of polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). Concentrations of PCNs were not correlated with PCB concentrations. The contribution of PCNs to dioxin-like toxic equivalents (TEQs) in human adipose tissues was estimated to be <1% of the polychlorinated dibenzo-p-dioxin/dibenzofuran (PCDD/F)-TEQs.     

Study on Fish and Amphibian Embryo-Larval Toxicity Test

In China, it is necessary to establish short-term biomonitoring methods for evaluating the chronic toxicity of industrial wastewater. In this paper, the aquatic toxicity of four important pollutants in petrochemical wastewater, including acrylonitrile, acetonitrile, sulphocyanic sodium and dimethyl formamide, was studied with fish and amphibian embryo-larval toxicity tests using Cyprinus carpio and Rana nigromaculata as test organisms. The objective is to present some useful data for establishing short-term chronic toxicity test methods used to perform biomonitoring in China. The results indicate that the chronic toxicity of acrylonitrile to fish and amphibians was the highest of the four test pollutants, followed by NaSCN, acetonitrile and DMF. The sensitivity of the carp embryo- larval test to chemicals was greater than that of the R. nigromaculata embryo-larval test.     

苯酚、苯胺及其衍生物对斜生栅列藻的急性毒性及QSAR研究

本文应用ＯＥＣＤ标准方法测定了１８种酚类胺类化合物对斜生栅列藻的４８ｈ－ＥＣ６０值，选择参数ＥＬＵＭＯ和ｌｇＫｏｗ进行了ＱＳＡＲ分析，得到下列回归方程：－１ｇＥＣ５０＝２．５４１－０．６４９ＥＬＵＭＯ＋０．２９９ＬｇＫｏｗ，ｎ＝１８，ｒ＝０．９０３，ＳＥ＝０．３１４应用所得方程计算了化合物的毒性，探讨了毒机理。     

1—硝基芘对斜生栅藻的毒性研究

酿要用评价化学品对藻类毒性的标准实验方法，研究了１－硝基芘对斜生栅藻生长的影响，得到了１－ＮＰ抑制斜生栅藻生长的ＥＣ５０（９６ｈ）为０．１８２４ｍｇ．１＾－１，实验结果还表明，１－ＮＰ对藻生长的抑制作用随时间的延长而增加，抑制物性表现为特久且强度大，抑制机理为不可逆性抑制。     

添加不同钝化剂降低污泥堆肥的植物毒性研究

高温堆肥后污泥的土地利用可充分发挥资源化、无害化的优势，污泥土地利用防止重金属污染一直是污泥施用中最重要的问题。通过测验发芽率指数这一代表植物毒性的指标证明，通过在污泥堆肥中加入粉煤灰、磷矿粉和草炭等不同的改性剂或重金属钝化剂可大大降低堆肥的植物毒性，从而降低污泥中重金属的可利用性。