Lysosomal,genetic and chromosomal damage in haemocytes of the freshwater bivalve (Unio pictorum) exposed to polluted sediments from the River Cecina (Italy)

The response to chemical disturbance of an Italian river (Cecina River) characterised by high levels of trace metals was assessed in the sentinel species freshwater painter’s mussel (Unio pictorum). We exposed U. pictorum in the laboratory to metal, metalloid and other trace element polluted river sediments in order to evaluate genetic and cellular biomarkers. Bivalves exposed to sediments taken from all the impacted sites of the river basin exhibited a significant impairment at the DNA, chromosomal and lysosomal levels. The lysosomal membrane stability of circulating haemocytes, assessed by the Neutral Red Retention Time, was found to be higher in specimens from Lake Maggiore and Berignone, while exhibited a significant decrease in specimens exposed to Ponteginori sediments. An increase of DNA migration in specimens exposed to the two contaminated sites in comparison with controls was found. Moreover, higher micronucleated haemocytes frequencies were observed in bivalves exposed to sediments collected at Possera 1 site. This study suggested that biomarkers evaluating cellular impairment, genetic and chromosomal damages, are highly sensitive and suitably applied to laboratory experiments. The painter’s mussel was confirmed to be an appropriate sentinel species for quality assessment in metal-contaminated freshwater environments.     

High-throughput screening of dioxins in sediment and soil using selective pressurized liquid extraction with immunochemical detection

A high-throughput screening method using selective pressurized liquid extraction (SPLE) and enzyme-linked immunosorbent assay (ELISA) for monitoring dioxins in sediment and soil is described. SPLE conditions were developed by extracting sediment or soil together with alumina, 10% AgNO₃ in silica, and sulfuric acid impregnated silica (acid silica) using dichloromethane (DCM) as the solvent at 100 °C and 2000 psi. Post-extraction cleanups were not required for ELISA. Two reference sediments (National Institute of Standards and Technology SRM 1944 and Wellington Laboratories WMS01) were analyzed by the SPLE–ELISA method. The ELISA utilized a polyclonal antibody and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as the calibrant. Recoveries of ELISA-derived TCDD equivalents (EQ) relative to the expected gas chromatography/high resolution mass spectrometry (GC/HRMS) derived dioxin toxic equivalent (TEQ) values were 116 ± 11% for SRM 1944 and 102 ± 13% for WMS01. ELISA TCDD EQs were consistent with the dioxin TEQs as measured by GC/HRMS for 25 soil/sediment samples from seven different contaminated sites. The ELISA had an approximate method detection limit of 10 pg g⁻¹ with a precision of 2.6–29% based on the relative percentage difference (%RPD) for duplicate samples. Estimated sample throughput for the SPLE–ELISA was three times or more than that of the GC/HRMS method employing PLE with a multi-column cleanup.     

Potential use of DNA adducts to detect mutagenic compounds in soil

In this study, three different soils with contrasting features, spiked with 300 mg benzo[a]pyrene (BaP)/kg dry soil, were incubated at 20 °C and 60% water holding capacity for 540 days. At different time points, BaP and DNA were extracted and quantified, and DNA adducts were quantified by ³²P-postlabelling. After 540 days incubation, 69.3, 81.6 and 83.2% of initial BaP added remained in Cruden Bay, Boyndie and Insch soils, respectively. Meanwhile, a significantly different amount of DNA-BaP adducts were found in the three soils exposed to BaP over time. The work demonstrates the concept that DNA adducts can be detected on DNA extracted from soil. Results suggest the technique is not able to directly reflect bioavailability of BaP transformation products. However, this new method provides a potential way to detect mutagenic compounds in contaminated soil and to assess the outcomes of soil remediation.     

二苯甲酮对斑点叉尾鮰鱼卵巢细胞生长的影响

试验通过直接、间接两种染毒手段来探讨二苯甲酮(benzophenone)对斑点叉尾鮰鱼卵巢细胞(CCO)生长的影响,以MTT法为手段测定染毒前后CCO细胞的存活数量和各阶段的生长状态,结果发现:当二苯甲酮以1～10μg/mL的浓度直接作用于CCO细胞时,CCO细胞生长在不同阶段均受到抑制作用:在指数增长期细胞生长速率减小,稳定期的存活量约为正常条件下的72%～91%,并且随着二苯甲酮浓度的增大,对CCO生长的抑制效应也相应的加剧;但是1μg/mL浓度的二苯甲酮在经过斑点叉尾鮰鱼原代肝细胞代谢后间接作用时,其代谢产物对CCO的生长在各阶段又呈现明显的促进作用,稳定期细胞数量约为正常条件下的109%。     

The detection of dioxin- and estrogen-like pollutants in marine and freshwater fishes cultivated in Pearl River Delta, China

In this study we aimed to assess the dioxin- and estrogen-like activities of contaminants extracted from twenty species of freshwater and seawater fishes, using luciferase reporter assays. Transfected MCF7 cells were treated with sample extracts and luciferase activities were then measured at 24-h of post-treatment. The mean values of the detected dioxin- and estrogen-like activities in the freshwater fishes were 25.3 pg TEQ/g ww and 102.3 pM EEQ/g ww whereas in the seawater fishes, the values were 46.2 pg TEQ/g ww and 118.8 pM EEQ/g ww. Using sample-relevant dosage of estrogen, inductions of cell proliferation markers (i.e. retinoblastoma, cyclin D) and stimulations of cell growth were revealed by Western blotting, colony formation and BrdU uptake assays. A cotreatment with TCDD significantly reduced these effects. Using the sample extracts with different dioxin- and estrogen-like activities, similar observation was revealed. The data highlighted the mixture effect of food contaminants on human health.     

Development of competitive immunoassays to hydroxyl containing fungicide metabolites

This paper describes the isolation of monoclonal antibodies and the development of competitive immunoassays to pesticide metabolites of the fungicides imazalil, carbendazim and thiabendazole. The metabolite specific hydroxyl residues were used as the reactive group with which to link the metabolite to the carrier proteins Keyhole Limpet Haemocyanin (KLH) and Bovine Serum Albumin (BSA). In each case immune responses in mice were raised and monoclonal antibodies were produced. Antibodies were developed into competitive ELISAs to the appropriate metabolite. The antibody raised to a metabolite of imazalil was optimised into a competitive ELISA format which had an assay IC50 of 7.5 μg/L and a limit of detection (LOD) of 1.1 μg/L. A single antibody isolated against the metabolite of carbendazim had assay IC50s of 3.2 and 2.7 μg/L for the metabolites of carbendazim and thiabendazole respectively with an LOD of 0.38 μg/L for both. These sensitive immunoassays may have application in the monitoring of human exposure to these fungicide residues either by occupational or non-occupational routes.     

Detection of environmental clastogens and aneugens in human fibroblasts by cytokinesis-blocked micronucleus assay associated with immunofluorescent staining of CENP-A in micronuclei

The cytokinesis-blocked micronucleus (CBMN) assay, in combination with fluorescent in situ hybridization (FISH) of human pan-centromeric DNA probes, or with CREST antibodies that specifically stain kinetochore proteins, is widely used on several cell types. It distinguishes micronuclei containing one or several whole chromosomes, which are positively labeled (centromere positive micronucleus, C+MN, due to aneugenic effect), or acentric chromosome fragments, which are unlabeled due to the absence of centromere (centromere negative micronucleus, C−MN, due to clastogenic effect). However, the very slight level of the centromeric signals obtained with the FISH technique on primary human fibroblasts, a cell type commonly used in environmental genetic toxicology, leads to great difficulties in distinguishing C+MN and C−MN. Furthermore, the CREST technique may lead to inappropriate results particularly with regards to variations in antibody composition between patient sera. Our results show that the in vitro CBMN, in combination with immunofluorescence staining of CENP-A (centromere protein A), efficiently screens genotoxicants for their ability to induce clastogenic and/or aneugenic effects. We propose the in vitro CBMN assay in combination with immunofluorescence staining of CENP-A as a suitable tool in environmental genotoxicity testing of primary human fibroblasts.     

Studies on the toxic effects of pentachlorophenol on the biological activity of anaerobic granular sludge

In order to explore the pathway of the anaerobic biotreatment of the wastewater containing pentachlorophenol (PCP) and ensure the normal operation of Upflow Anaerobic Sludge Blanket (UASB) reactor, the anaerobic sludge under different acclimation conditions were selected to seed and start up UASB reactors. Anaerobic toxicity assays were employed to study the biological activity, the tolerance and the capacity to degrade PCP of different anaerobic granular sludge from UASB reactors. Results showed that the anaerobic granular sludge acclimated to chlorophenols (CPs) could degrade PCP more quickly (up to 9.50mg-PCPg(-1)TVSd(-1)). And the anaerobic granular sludge without acclimation to CPs had only a little activity of degrading PCP (less than 0.07mg-PCPg(-1)TVSd(-1)). Different PCP concentrations (2, 4, 6, 8mgL(-1)) had different inhibition effects on glucose utilization, volatile fatted acidity (VFA)-degrading and methanogens activity of PCP degradation anaerobic granular sludge, and the biological activity declined with the increase in PCP concentration. The methanogens activity suffered inhibition from PCP more easily. The different acclimation patterns of seeded sludge had distinctly different effects on biological activity of the degradation of PCP of anaerobic granular sludge from UASB reactors. The biological activity of the anaerobic granular sludge acclimated to PCP only was also inhibited. This inhibition was weak compared to that of anaerobic granular sludge acclimated to CPs, further, the activity could recover more quickly in this case. In the same reactor, the anaerobic granular sludge from the mid and base layers showed higher tolerance to PCP than that from super layer or if the sludge is unacclimated to CPs, and the corresponding recovery time of the biological activity in the mid and base layers were short. Acetate-utilizing methanogens and syntrophic propinate degraders were sensitive to PCP, compared to syntrophic butyrate degraders.     

Evaluation of the fish short term reproduction assay for detecting endocrine disrupters

In a fish testing strategy, positive results of the fish short term reproduction assay (FSTR), often trigger a definitive test like the fish sexual development test (FSDT) or the fish full life cycle test (FFLC), entailing ethical and economic problems. This study analysed 137 studies encompassing 35 chemicals with different modes of actions (MOAs). Variability is quantified for MOA endpoints vitellogenin (VTG) and secondary sex characteristics (SSCs) as well as for apical endpoints. Two MOA endpoints could indicate estrogenic, anti-estrogenic, androgenic, anti-androgenic and steroidogenesis activities. Great variability, however, has been observed for chemicals with anti-androgenic and steroidogenesis activities, suggesting that TG229/230 may not be sensitive enough to detect these types of chemicals and may produce false negatives. Changes in apical endpoints like fecundity are not limited to endocrine disrupting chemicals (EDCs). Non-EDCs could induce the similar effects on these apical endpoints. If elucidating MOA is needed, targeted in vitro MOA tests are suggested. Positive in vitro MOA results trigger a definitive test, which could be used for confirmation of the MOA in vivo and for deriving a no observed effect concentration (NOEC). Based on positive MOA results of TG229, a definitive test such as the FSDT or the FFLC is still needed, because the current TG229 has limitation on the derivation of a NOEC. An extended TG229 with more power to detect reproduction effects, as recently proposed in the OECD test guideline program, would improve the possibility to derive a NOEC and increase its usefulness in risk assessment.     

Immunoassay of red dyes based on the monoclonal antibody of β-naphthol

The objective of the present study was to develop a multi-analyte immunoassay for the determination of eight red dyes in food samples. Two dye intermediates (2-hydroxy-1-naphthoic acid and 1-amino-2-naphthol) were used as the haptens to produce the monoclonal antibodies. The obtained monoclonal antibodies recognized Sudan 1–4, Para red, Sudan red G, Sudan red B and Acid orange II simultaneously. After evaluation of different antibody/coating antigen combinations, a heterologous indirect competitive enzyme linked immunosorbent assay was developed to determine the eight red dyes in food samples (chili oil, chili powder, tomato sauce, hotpot seasoning). The crossreactivities to the eight analytes were in the range of 61%–79% (with β-naphthol as 100%), and the limits of detection were in the range of 1.3–1.9 ng/mL. The recoveries of the eight analytes from the fortified blank samples were in the range of 84.2%–115% with coefficients of variation lower than 18.3%. Therefore, this method could be used as a rapid and simple tool to detect the residues of the eight red dyes in foods.