Cadmium and Lead Levels in Fish (Tilapia Nilotica) Tissues as Biological Indicator for Lake Water Pollution

Cadmium and lead were determined in different tissues (muscle,gill, stomach, intestine, liver, vertebral column and scales) of Tilapia nilotica from the High Dam Lake, Aswan (Egypt) to assess the lake water pollution with those toxic metals. Fish samples were chosen from different ages and weights to be analyzed along with samples of the aquatic plant(Najas armeta), sediment and lake water.The results showed that cadmium and lead concentrations were higher in fish scales and vertebral column than in the other parts of the fish. Cadmium and lead levels in High Dam lake water and fish (Tilapia nilotica) were a result of the pollution which uptakes from aquatic plants, sediments andgasoline containing lead that leaks from fishery boats. Tilapia nilotica fish was used as a good bio-assay indicator for the lake pollution with cadmium and lead. The fish musclesin this study were in the safety baseline levels for man consumption.     

武汉东湖湖水的藻类生长潜力(AGP)测试

在东湖湖水样品中添加排入东湖的主要污水或营养物(氮和磷)进行藻类测试,观察它们对斜生橱藻(Scenedesmus obliquus)的生长促进作用.生长反应与添加的污水浓度成正比,其SC_(20)(促进20％增长的浓度)为0.5—4％.单独添加氮或磷,在高浓度情况下也很少促进藻类生长,但共同添加时大多有促进作用.东湖为一严重富营养型湖泊,为了控制其富营养化进程,污水截流应是首先要采取的一项措施.     

Modulation of steroidogenesis by coastal waters and sewage effluents of Hong Kong, China, using the H295R assay

BACKGROUND, AIM, AND SCOPE: The presence of a variety of pollutants in the aquatic environment that can potentially interfere with the production of sex steroid hormones in wildlife and humans has been of increasing concern. The aim of the present study was to investigate the effects of extracts from Hong Kong marine waters, and influents and effluents from wastewater treatment plants on steroidogenesis using the H295R cell bioassay. After exposing H295R cells to extracts of water, the expression of four steroidogenic genes and the production of three steroid hormones were measured. MATERIALS AND METHODS: Water samples were collected during the summer of 2005 from 24 coastal marine areas and from the influents and effluents of two major waste water treatment plants (WWTPs) in Hong Kong, China. Samples were extracted by solid phase extraction (SPE). H295R cells were exposed for 48 h to dilutions of these extracts. Modulations of the expression of the steroidogenic genes CYP19, CYP17, 3betaHSD2, and CYP11beta2 were determined by measuring mRNA concentrations by real-time polymerase chain reaction (Q-RT-PCR). Production of the hormones progesterone (P), estradiol (E2), and testosterone (T) was quantified using enzyme linked immunosorbent assays (ELISA). RESULTS: Extracts from samples collected in two fish culture areas inhibited growth and proliferation of H295R cells at concentrations greater or equal to 10(5) L equivalents. The cells were exposed to the equivalent concentration of active substances in 10,000 L of water. Thus, to observe the same level of effect as observed in vitro on aquatic organisms would require a bioaccumulation factor of this same magnitude. None of the other 22 marine samples affected growth of the cells at any dilution tested. Twelve of the marine water samples completely inhibited the expression of CYP19 without affecting E2 production; inhibition of CYP17 expression was observed only in one of the samples while expression of CYP11beta2 was induced as much as five- and ninefold after exposure of cells to extracts from two locations. The expression of the progesterone gene 3betaHSD2 was not affected by any of the samples; only one sample induced approximately fourfold the production of E2. Although more than twofold inductions were observed for P and T production, none of these values were statistically significant to conclude effects on the production of these two hormones. While influents from WWTPs did not affect gene expression, an approximately 30% inhibition in the production of E2 and a 40% increase in P occurred for the exposure with influents from the Sha Tin and Stonecutters WWTPs, respectively. Effluents from WWTPs did not affect the production of any of the studied hormones, but a decrement in the expression of the aldosterone gene CYP11beta2 was observed for the Sha Tin WWTP exposure. No direct correlation could be established between gene expression and hormone production. DISCUSSION: Observed cytotoxicity in the two samples from fish culture areas suggest the presence of toxic compounds; chemical analysis is required for their full identification. Although effluents from WWTPs did not affect hormone production, other types of endocrine activity such as receptor-mediated effects cannot be ruled out. Interactions due to the complexity of the samples and alternative steroidogenic pathways might explain the lack of correlation between gene expression and hormone production results. CONCLUSIONS: Changes observed in gene expression and hormone production suggest the presence in Hong Kong coastal waters of pollutants with endocrine disruption potential and others of significant toxic effects. The aromatase and aldosterone genes seem to be the most affected by the exposures, while E2 and P are the hormones with more significant changes observed. Results also suggest effectiveness in the removing of compounds with endocrine activity by the WWTPs studied, as effluent samples did not significantly affect hormone production. The H295R cell showed to be a valuable toll in the battery required for the analysis of endocrine disrupting activities of complex environmental samples. RECOMMENDATIONS AND PERSPECTIVES: Due to the intrinsic complexity of environmental samples, a combination of analytical tools is required to realistically assess environmental conditions, especially in aquatic systems. In the evaluation of endocrine disrupting activities, the H295R cell bioassay should be used in combination with other genomic, biological, chemical, and hydrological tests to establish viable modes for endocrine disruption and identify compounds responsible for the observed effects.     

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)     

Physicochemical substance properties as indicators for unreliable exposure in microplate-based bioassays

In the last years many efforts were made to transform standardized algal test protocols into low-cost microplate assays. While advantages were pointed out frequently, limitations are not systematically addressed, thus hindering a widespread utilisation. In this study a group of organic substances with a wide distribution of volatility (log K_AW from −6.53 to −2.13) and lipophilicity (log K_OW from 1.26 to 4.92) was investigated with respect to the influence of these physicochemical properties on their algal toxicity in different assays. Therefore the EC₅₀ values were determined with a microplate assay based on ISO 8692 protocol and the results were compared with those of an established algal growth inhibition test conducted in air tight glass vessels. Using the ratio of the EC₅₀ values, a clear connection between biological response and volatility as well as lipophilicity of test substances could be detected. Chemicals with a log K_OW higher than 3 or a Henry coefficient log K_AW higher than −4 were identified as less effective in the microplate assay than in the comparative assay. The loss in nominal concentration due to physicochemical properties could be shown to contribute to this using HPLC analysis. Consequently, when using microplate assay’s one should be aware that lipophilic and volatile chemicals might be underestimated in their toxicity, which could be indicated from evaluating related physicochemical properties modelled from structural information prior to an experimental investigation.     

Evaluation of Caging Designs and a Fingernail Clam for Use in an In Situ Bioassay

Two cage designs and fingernail clams(Sphaerium fabale) were evaluated for theirsuitability for use in in situ bioassays toassess the ecological condition of a stream andpredict ecological recovery potential. One design(referred to as tray design) was a modified plastictray about one-fourth full of small gravels andcovered with 1 mm fiberglass mesh. The second design(referred to as tube-plates) consisted of shortplexiglass tubes about one-third full of small gravelsand attached horizontally to a plexiglass plate. Oneend of each tube faced into the current; both endswere covered with mesh. Cages containing clams weredeployed at reference and impacted (test) sites forperiods of 70 to 135 d. Growth and survival were theprimary endpoints evaluated, but the tube-platesallowed isolation of individual clams so that natalityalso could be evaluated as an endpoint. Results ofbenthic macroinvertebrate surveys, performed foranother study, were included to help validate bioassayresults. Both cage designs yielded good quantitative,site-specific results for clam survival and growth;results for natality, though, were less conclusive. Clam survival and growth results were in good generalagreement with the results for the benthicmacroinvertebrate community surveys. At a site wherethe macroinvertebrate community was the mostdepauperate, clam mortality was always rapid. At asite where the condition of the macroinvertebratecommunity was only slightly less impacted than themost impacted site, clam growth was almost alwayssignificantly lower than at reference sites. Survivalof clams was significantly reduced in <25 d at thissite in some trials, but in other trials there waslittle mortality. At a minimally impacted site, clamsurvival was similar to that found at reference sites,and differences in clam growth were not detectableuntil after 40 to 50 d of exposure. The tube-platedesign was easier to use, allowed more flexibility inselection of response parameters, and required lesshandling time of test animals, thus, this was thepreferred design. Our results demonstrated thateither in situ bioassay design can be used toaugment monitoring and assessment programs. Their useas a predictor of ecological recovery, however,requires further evaluation.     

Cultured human-cell-based bioassay for environmental risk management

Among bioassays for evaluating various impacts of chemicalson humans and ecosystems, those based on culturedmammalian-cells can best predict acute lethal toxicity to humans. Weexpect them to be employed in the future in environmentalrisk management alongside mutagenicity tests and endocrine-disrupting activity tests. We recently developed adisposable bioassay device that immobilizes humanhepatocarcinoma cells in a small micropipette tip. Thisenables very quick (within 2 h) evaluation of acute lethaltoxicity to humans. For bioassay-based environmentalmanagement, 2 promising approaches have been demonstrated bythe US-EPA: toxicity identification evaluation (TIE) andtoxicity reduction evaluation (TRE). The Japanese Ministryof Environment has been supporting a multi-center validationproject, aimed at assembling a bioassay database. To makefull use of these resources, we present a numerical modelthat describes contribution of individual chemical toobserved toxicity. This will allow the selection of the mosteffective countermeasure to reduce the toxicity. Bioassay-based environmental risk management works retrospectively,whereas impact assessment using substance flow models andtoxicity databases works prospective. We expect that these 2approaches will exchange information, act complementarily,and work effectively in keeping our environment healthy inthe 21st century.     

CADMIUM ACCUMULATION AND TOXICITY IN SUBMERGED PLANT HYDRILLA VERTICILLATA (l.f.) ROYLE

Cadmium accumulation and its toxicity in relation tochlorophyll, protein, cysteine contents and in vivo nitrate reductaseactivity were studied under controlled conditions in Hydrillaverticillata, a submerged commonly occurring macrophyte. Plants weresubjected to six different concentrations of Cd ranging from 1.0 to 25.0 µM for 24, 48, 72 and 168 h. Tissue Cd concentration was maximum (13.71 µmoles/g dw) at 25 µM background concentration. At this concentration, a decrease of approximately 79 and 72% was found in chlorophyll and protein content. In vivo nitrate reductase activity was stimulated at 1.0 µM; however, the activity gradually declined beyond this concentration. Exposure to various cadmium concentrations resulted in an increase in cysteine content of the plant.     

Determination of Toxic Pollutants in Water Using A Marine Phytoplankton Dunaliella Bioculata and Doppler Laser Velocimetry

This paper reports an experimental study of the response of the unicellular flagellated algae Dunaliella bioculata to toxic pollutants in water using the 'Doppler laser velocimetry' method. the response to toxic pollutants was encouraging and indicates that Dunaliella bioculata is a good biological model for the detection of toxic pollutants in water.