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)     

Multiresidue analysis of pesticides in fruits and vegetables by gas chromatography-mass spectrometry

A multiresidue method was assessed for the determination of several pesticides (organochlorine, organophosphorus, pyrethroids, triazole, amidine) using gas chromatography/mass spectrometry. The extraction of pesticides was carried out by liquid-liquid extraction (LLE) and solid-phase extraction (SPE) using two types of columns (CN and C18). The extracts were cleaned by the addition of florisil, the pesticides were separated by capillary column gas chromatography and detected by mass spectrometry in the electron impact mode. The extraction using C18 column provided the best results for most of the analyzed pesticides. The majority of pesticides recoveries from the four fruits and vegetables (apples, pears, tomatoes and pepper) were greater than 60%. Linearity and precision were satisfactory. The estimated limits of detection and limits of quantification ranged from 0.01 to 0.1 mg/kg and from 0.02 to 0.3 mg/kg, respectively. The proposed procedure was found to be useful for the multiresidue analyses of pesticides in agricultural products for routine monitoring programs.     

Comparison of two metal surveys by moss Tortula ruralis in Budapest, Hungary

Moss transplants of Tortula ruralis were used as active biomonitoring organisms as part of a monitoring study to assess the metals (Al, Cd, Cu, Cr, Fe, Ni, Pb, V and Zn) associated with ambient particles on mosses in Budapest, the capital town of Hungary. The moss samples were collected in a nature conservation area from a semi-arid sand grassland (Festucetum vaginatae danubiale), less than 1 month before transplantation. Moss cushions were exposed to pollution in Budapest during October–November 1993 and February–March 1994. In the study area, 16 sites were marked out as measuring sites, in accordance with the structure of the city. A similar stand was established in the Botanical Gardens of the Szent István University in Gödöll? as a control site. ICP-AES analysis of moss for metals showed the ability of Tortula ruralis to accumulate the metals under study. Control site showed lower impact in comparison to the other sites.     

The Distribution and Sources of Polycyclic Aromatic Hydrocarbons in Surface Sediments Along the Egyptian Mediterranean Coast

Coastal marine sediment samples were collected from 31 sampling stations along the Egyptian Mediterranean Sea coast. All sediment samples were analyzed to determine aliphatic and polycyclic aromatic hydrocarbons (PAHs) as well as total organic carbon (TOC) contents and grain size analysis. Total concentrations of 16 EPA-PAHs in the sediments were varied from 88 to 6338 ng g⁻¹ with an average value of 154 ng g⁻¹ (dry weight). However, the concentrations of total aliphatic were varied from 1.3 to 69.9 ng g⁻¹ with an average value of 15.6 ng g⁻¹ (dry weight). The highest contents of PAHs were found in the Eastern harbor (6338 ng g⁻¹), Manzala (5206 ng g⁻¹) and El-Jamil East (4895 ng g⁻¹) locations. Good correlations observed between a certain numbers of PAH concentrations allowed to identify its origin. The average total organic carbon (TOC) percent was varied from 0.91 to 4.54%. Higher concentration of total pyrolytic hydrocarbons (∑COMB) than total fossil hydrocarbons (∑PHE) declared that atmospheric fall-out is the significant source of PAHs to marine sediments of the Egyptian Mediterranean coast. The selected marked compounds, a principal component analysis (PCA) and special PAHs compound ratios (phenanthrene/anthracene vs fluoranthene/pyrene; ∑COMB/∑EPA-PAHs) suggest the pyrogenic origins, especially traffic exhausts, are the dominant sources of PAHs in most locations. Interferences of rather petrogenic and pyrolytic PAH contaminations were noticed in the harbors due to petroleum products deliveries and fuel combustion emissions from the ships staying alongside the quays.     

Isolation and Identification of Enterococci from Seawater Samples: Assessment of Their Resistance to Antibiotics and Heavy Metals

A hundred Enterococcus strains were isolated from seawater samples collected from coastal areas of Istanbul. Isolates were identified to the species level using standard biochemical tests specified by Facklam and Collins. The species distribution was as follows Enterococcus faecalis (96%), Enterococcus gallinarum (3%) and Enterococcus solitarius (1%). The resistance of bacteria to both heavy metals (zinc [Zn], iron [Fe], cadmium [Cd], chrome [Cr], cobalt [Co]) and antibiotics (ampicillin 10 μg [AP], penicillin G 10 Units [PG], gentamycin 10 μg [GM], streptomycin 10 μg [S], chloramphenicol 10 μg [C], erythromycin 15 μg [E], kanamycin 30 μg [K], amikacin 30 μg [AK], nalidixic acid 30 μg [NA], and vancomycin 30 μg [VA]) was evaluated. None of the strains was resistant to VA. It was found that among the 100 isolates, those that exhibit resistance to antibiotics, particularly NA, S and K, were also resistant all the heavy metals tested. To our knowledge this is the first report focusing on determination of resistance of environmental enterococci found in Istanbul against heavy metals and antibiotics. Thus, combined expressions of antibiotic and heavy metal resistance may help to reinforce ecological and epidemiological studies and to determine the role of these strains in antibiotic and heavy metal resistance dissemination.     

Application of a Larval Medaka Assay to Evaluate the Fish Safety Level in Sagami River, Japan

Physico-chemical characteristics of some river and hand-dug well waters used for drinking and domestic purposes in the oil rich Niger Delta area of Nigeria were assessed using standard methods. The concentrations of the parameters in the river water samples ranged in the following order: pH (5.6–6.9), temperature (26.90–28.60°C), turbidity (23–63 NTU), electrical conductivity (52–184 μs/cm), DO (5.4–7.2 mg/l), BOD (21–57 mg/l), TDS (6.0–217 mg/l), PO₄ ³⁻ (0.19–1.72 mg/l), SO₄ ²⁻ (25–36.8 mg/l), NO₃ ⁻ (20.3–28 mg/l), Fe (6.07–15.71 mg/l), Zn (0.04–0.24 mg/l), Pb (0.01–0.17 mg/l), Ni (0.01–0.13 mg/l), Vn (0.01–0.20 mg/l) and Hg (0.001–0.002 mg/l). The concentrations of these parameters in the hand-dug well water ranged in the following order: pH (5.7–6.8) temperature (26–30°C), turbidity (134–171 NTU), electrical conductivity (160–340 μs/cm), DO (5.4–6.4 mg/l), BOD (13–34 mg/l), TDS (110–190 mg/l), PO₄ ³⁻ (0.84–1.84 mg/l), SO₄ ²⁻ (10.6–28.1 mg/l), NO₃ ⁻ (11.3–23 mg/l), Fe (13.17–16.31 mg/l), Ni (0.01–0.02 mg/l), Vn (0.01–0.04 mg/l) and Hg (0.001–0.004 mg/l). The concentrations of BOD, turbidity, NO₃ ⁻ and Fe in the water samples were above WHO and FMENV permissible limits for safe drinking water. The results suggest that the use of such waters for drinking and domestic purposes pose a serious threat to the health of the users and calls for the intervention of government agencies.     

The Influence of a Coal-Fired Power Plant in Turkey on the Chemical Composition of Rain Water in a Certain Region

In this study, the chemical composition of the rainwater in Yatağan, which is a region surrounding a coal power plant was investigated from February to April 2002. Rainwater samples were obtained from Yatağan, located northwest of Muğla City in Turkey. pH values and concentrations of major ions (Ca²⁺, Na⁺, K⁺, , , ) in the rainwater samples were analyzed. The pH varied from 5.1 to 7.9 with an average of 6.7 which was in alkaline range considering 5.6 as the neutral pH of cloud water in equilibrium with atmospheric CO₂. In the total of 30 rain events, only three events were observed with water in the acidic range (<5.6), which occurred after continuous rains. The equivalent concentration of components followed the order: Ca²⁺ > > Na⁺ > > > K⁺ > H⁺. The anion and cation concentrations in the rainwater samples showed a high sulphate concentration (131 μEq/l), as well as high sodium (40 μEq/l) and calcium (298 μEq/l) concentrations. These values indicate that one probable source of the high sodium concentration is fly ash, after the coal burning process and the power plant can be effective on level concentrations in rainwater. In addition, the dust-rich local and surrounding limestone environment might have caused the high concentration of Ca²⁺ in rainwater of the Yatağan Basin. Due to a large contribution of these cations to the sulphate neutralization action, the rainwater of this region displays only a moderate acidity, which does not cause significant environmental impact.     

Ozone pre-treatment as improver of PAH removal during anaerobic digestion of urban sludge

Polycyclic aromatic hydrocarbons are ubiquitous persistent pollutants. They may accumulate in sludge during wastewater treatment because of their low biodegradability and their hydrophobic characteristics. Combination of ozonation and anaerobic digestion may be efficient to remove PAHs naturally present in sludge. The objective of this study was to investigate the impact of ozone pre-treatment, with and without surfactant addition, on the anaerobic degradation of 12 PAHs (from low to high molecular weight). Under anaerobic digestion without ozonation pre-treatment, the highest removals were obtained for the lightest PAHs (3-aromatic rings). Ozonation pre-treatment of sludge allowed to increase biodegradability or bioavailability of each PAH, and the PAH removals were well correlated to the PAH solubility. Finally, addition of tyloxapol before sludge ozone pre-treatment had antagonist effects on PAH removal during anaerobic digestion: negative impact on anaerobic ecosystem activity and improvement of PAH bioaccessibility (particularly the PAHs with the highest octanol water partition coefficients).     

Optimisation of pressurised liquid extraction for elimination of sulphur interferences during determination of organotin compounds in sulphur-rich sediments by gas chromatography with flame photometric detection

A simple method for species-selective analysis of organotin compounds (OTCs) (butyl and phenyl) in sediments was developed. The sample preparation procedure was specifically optimised for sulphur-rich sediments to eliminate interferences from elemental sulphur and organosulphur compounds. Tin species were extracted from sediment samples using pressurised liquid extraction technique (PLE), ethylated - with simultaneous extraction to isooctane - in aqueous phase with sodium tetraethylborate (NaBEt(4)) and separated/detected by gas chromatography with flame photometric detection (GC-FPD). PLE operational variables (extraction temperature and pressure, solvent composition and number of static extraction steps) and extract handling routine were fine-tuned to minimise the amount of extracted interferents while keeping OTCs recovery at an acceptable level. Best results were obtained after extraction of sediment samples with methanol/water (75% v/v methanol) solution of acetic acid/sodium acetate with tropolone addition (0.6 g l(-1)). Derivatisation of low temperature, high-pressure (50 degrees C, 13.8 MPa) extracts gives isooctane extracts which are clean enough to be directly analysed by GC-FPD without any further cleanup. Interferences from elemental sulphur were completely eliminated while concentrations of other interferents were reduced to the level not impairing quantitation of OTCs under the study. No negative effects in terms of chromatographic column deterioration were observed after repeated injections of such extracts. Two certified reference materials, BCR646 and PACS-2, were analysed to assess performance of the method. Recoveries of all OTCs under the study, except MBT, were in the range of 91-114%. MBT extraction efficiency was low (34-47%) therefore the method is unsuitable for precise determinations of this compound.     

High levels of organochlorines in mothers' milk from Chennai (Madras) city, India

Mothers' milk from Chennai (formerly Madras), India and three other places Perungudi, the municipal dumping site of south Chennai area (situated at the suburb of Chennai), Chidambaram, a predominantly agricultural town situated 250 km south of Chennai and Parangipettai, a fishing village 15 km north of Chidambaram, all situated at or near the southeastern Bay of Bengal coast of India were found to contain measurable concentrations of HCHs, DDTs, PCBs, CHLs and HCB. A notable finding in this study is that Chennai mothers have higher levels of HCHs in their milk and hence may transfer considerably higher amounts of the chemical than the mothers from all the other three places of the present study indicating a higher health risk to Chennai's children. It was also found that the levels of the two organochlorine pesticides (HCHs and DDTs) increased in Chennai mothers' milk in the last decade. Food items collected from Chennai markets did not show any remarkably higher levels of any of the chemicals measured in this study. Levels of the two classical organochlorines (DDTs and HCHs) have declined in many of the food items when compared with our data collected two decades before in the same locations, showing the effectiveness of the recent ban on both these chemicals in the country. The sources, possible health risks and the ways to curtail the effects of HCHs, especially at Chennai, should be investigated further.