Jette Rank

Genotoxicity testing of wastewater sludge using the Allium cepa anaphase-telophase chromosome aberration assay.

Wastewater sludges were analysed in the Allium cepa genotoxicity test. They were sampled during three winter periods from three Danish municipal wastewater treatment plants differing in size and industrial load. The toxicity of the sludge was tested in the Allium root inhibition assay, and the results expressed as EC30 and EC50 values showed that the toxicity could be positive correlated to the industrial load. However, when genotoxicity was tested at concentrations corresponding to the EC30 and EC50 values in the A. cepa anaphase-telophase assay, only two sludge samples from the smallest plant with the lowest industrial load induced significant chromosome aberrations. Concentrations of the heavy metals Pb, Ni, Cr, Zn, Cu, and Cd were also determined and could partly be correlated with the toxicity of the sludge and the industrial load of the treatment plants.

Allium cepa anaphase–telophase root tip chromosome aberration assay on N-methyl-N-nitrosourea, maleic hydrazide, sodium azide, and ethyl methanesulfonate

The Allium anaphase-telophase assay was used to show genotoxicity of N-methyl-N-nitrosourea (MNU), maleic hydrazide (MH), sodium azide (NaN3) and ethyl methanesulfonate (EMS). All agents induced chromosome aberrations at statistically significant levels. The rank of the lowest doses with positive effect was as follows: NaN3 0.3 mg/l < MH 1 mg/l < MNU 41 mg/l < EMS 100 mg/l. The results were compared with results from other plant assays (Arabidopsis, Vicia, Tradescantia) and for MH and MNU the values were found to be within the same range, whereas the results in the Allium test for NaN3 and EMS were in a lower range than that found for the other plant assays. EMS and MMS (methyl methanesulfonate), two chemicals used as positive controls in mutagenicity testing, were compared in the Allium test, and MMS was found to be about ten times more potent in inducing chromosome aberrations than EMS. Recording of micronuclei in interphase cells showed that this endpoint does not give more information of clastogenicity than recording of chromosome aberrations in anaphase-telophase cells.

Comet assay on gill cells and hemocytes from the blue mussel Mytilus edulis

Gill cells and hemocytes from the blue mussel Mytilus edulis were examined for DNA damage using the comet assay after laboratory exposure in vitro and in vivo to methyl methansulfonate (MMS). Hydrogen peroxide and UV radiation were used as positive control. Comet assay was also carried out on hemocytes from blue mussels sampled at polluted and unpolluted coastal areas. After 60 min in vitro exposure of gill cells to MMS, the highest response, a tail moment of 6.70+/-4.25, was obtained at 1.0mg/L. At higher doses the response decreased. After 2 days in vivo exposure a dose response was seen at concentrations between 1.0 and 33.0mg/L MMS for both gill cells and hemocytes. However, after 4 days in vivo exposure using the same concentrations of MMS, a maximum effect was seen at a 10 times lower concentration of 3.3mg/L. At the higher doses, the effect decreased. Hemocytes from blue mussels sampled at four polluted sites in Køge Bay had a great variation in tail moments with the highest value of 5.38+/-4.39. The average of all samples from Køge Bay had tail moments of 2.75+/-1.00(n=19), which was significantly higher (P<0.05) than the average, 1.72+/-1.16(n=10), of samples from unpolluted coastal waters.

On the Use of Mixture Toxicity Assessment in REACH and the Water Framework Directive: A Review

ABSTRACT This review seeks to connect the scientific theory of mixture toxicity to its implementation within different regulatory frameworks. The aim is to demonstrate how mixture toxicity assessment can be more thoroughly integrated into the European chemical regulations, REACH, and the Water Framework Directive (WFD), using the experiences gained through other regulatory frameworks. The article consists of (1) an examination of the scientific underpinnings of the common mixture toxicity assessment methods; (2) a discussion of how these methods have been used in regulatory frameworks; and (3) a discussion of how the methods could be applied within REACH and the WFD. It is concluded that concentration addition should be applied as a default model for mixture toxicity assessment. Furthermore, it is concluded that REACH and the WFD only include mixture toxicity assessments in specific situations. However, it is shown that it is scientifically feasible and regulatorally practicable to integrate a more holistic mixture toxicity approach into both legislations. In this connection, the experience gained from the U.S. frameworks on mixture toxicity assessment could be useful. The construction of a database that includes data on chemicals in the European environment could be used for mixture toxicity assessment of the chemicals with individual PEC/PNECs > 0.1.

Intersex in Littorina littorea and DNA damage in Mytilus edulis as indicators of harbour pollution.

Intersex in snails (Littorina littorea) and DNA damage in blue mussels (Mytilus edulis) were analysed to assess how these bio-indicators reflected the level of chemical contamination at two sites in a highly contaminated harbour in Denmark. The comet assay using mussel gill cells was an indicator of exposure to genotoxic chemicals, and the intersex index (ISI) observed in snails was an indicator of exposure to butyltin (BT) compounds. Biota and sediments were analysed for heavy metals (Cd, Cu, Pb and Zn), butyltin compounds (TBT, DBT and MBT), nine PCB congeners and 19 PAH compounds. The biological effects were found to reflect the levels of the chemicals, and it was concluded that intersex in L. littorea and DNA damage in M. edulis can be used as bio-indicators of harbour pollution.

Comet assay on tetraploid yeast cells.

Tetraploid yeast cells (Saccharomyces cerevisiae) were used in the comet assay with the intention of developing a new, fast and easy assay for detecting environmental genotoxic agents without using higher organisms. Two DNA-damaging chemicals, H(2)O(2) and acrylamide, together with wastewater from three municipal treatment plants were tested for their effect on the yeast-cell DNA. The main problem with using yeast in the comet assay is the necessity to degrade the cell wall. This was achieved by using Zymolase 100 T twice during the procedure, since Zymolase 20 T did not open the cell wall. Analytical problems that arose due to the small amount of DNA in the yeast nuclei in haploid and diploid cells, which contain 13 Mbp and 26 Mbp DNA per cell, respectively, were solved by using tetraploid yeast cells (52 Mbp) instead. DNA damage was shown after exposure to H(2)O(2) and acrylamide. The lowest dose causing significant DNA damage was 20 microM for H(2)O(2) and 200mg/l for acrylamide. Tertiary-treated wastewater from the outlets of three municipal wastewater-treatment plants was tested, but did not cause DNA damage. Even though it is possible to produce comets with tetraploid yeast cells, the amount of DNA is likely too small for a proper comet assay.

Genotoxic potential of two herbicides and their active ingredients assessed with comet assay on a fish cell line, epithelioma papillosum cyprini (EPC).

The aim of this study was to optimize the epithelioma papillosum cyprini (EPC) cell line handling procedure for the comet assay to investigate the genotoxic potential of widely used pesticides. The effects of various media and handling of the EPC cell line were examined. Results indicated that avoiding trypsin to detach cells led to lower level of DNA damage in the negative control. Further, two commonly used herbicides (Dezormon and Optica trio) and their four active ingredients (4-chloro-o-tolyloxyacetic acid, 2,4-dichlorophenoxyacetic acid, 2-(4-chloro-2-methylphenoxy)propionic acid, 2-(2,4-dichlorophenoxy)propionic acid) individually and in a ternary mixture were examined with the comet assay. Data showed that among the active ingredients only 2,4-D and MCPA induced DNA damage, while both herbicides were genotoxic at high concentrations.

Mixture Genotoxicity of 2,4-Dichlorophenoxyacetic Acid, Acrylamide, and Maleic Hydrazide on Human Caco-2 Cells Assessed with Comet Assay

Assessment of genotoxic properties of chemicals is mainly conducted only for single chemicals, without taking mixture genotoxic effects into consideration. The current study assessed mixture effects of the three known genotoxic chemicals, 2,4-dichlorophenoxyacetic acid (2,4-D), acrylamide (AA), and maleic hydrazide (MH), in an experiment with a fixed ratio design setup. The genotoxic effects were assessed with the single-cell gel electrophoresis assay (comet assay) for both single chemicals and the ternary mixture. The concentration ranges used were 0–1.4, 0–20, and 0–37.7 mM for 2,4-D, AA, and MH, respectively. Mixture toxicity was tested with a fixed ratio design at a 10:23:77% ratio for 2.4-D:AA:MH. Results indicated that the three chemicals yielded a synergistic mixture effect. It is not clear which mechanisms are responsible for this interaction. A few possible interactions are discussed, but further investigations including in vivo studies are needed to clarify how important these more-than-additive effects are for risk assessment.