Maria Näslund

An evaluation of free water surface wetlands as tertiary sewage water treatment of micro-pollutants.

Increased attention is currently directed towards potential negative effects of pharmaceuticals and other micro-pollutants discharged into the aquatic environment via municipal sewage water. A number of additional treatment technologies, such as ozonation, have therefore been suggested as promising tools for improving the removal efficiency of pharmaceuticals in existing Sewage Treatment Plants (STPs). Constructed wetlands are also capable of removing a variety of micro-pollutants, including some pharmaceuticals, and could hence be a resource efficient complement to more advanced treatment technologies. The purpose of the present study was therefore to increase the knowledge base concerning the potential use of constructed wetlands as a treatment step to reduce emissions of organic micro-pollutants from municipal sewage effluents. Under cold winter conditions, incoming and outgoing waters from four Swedish free water surface wetlands, operated as final treatment steps of sewage effluent from municipal STPs, were sampled and analyzed for levels of a set of 92 pharmaceuticals and 22 inorganic components as well as assessed using subchronic ecotoxicity tests with a macro-alga and a crustacean. Sixty-five pharmaceuticals were detected in the range from 1 ng L(-1) to 7.6 μg L(-1) in incoming and outgoing waters from the four investigated wetlands. Although the sampling design used in the present study lacks the robustness of volume proportional to 24h composite samples, the average estimated removal rates ranged from 42% to 52%, which correlates to previous published values. The effects observed in the ecotoxicity tests with the macro-alga (EC(50)s in the range of 7.5-46%) and the crustacean (LOECs in the range of 11.25-90%) could not be assigned to either pharmaceutical residues or metals, but in general showed that these treatment facilities release water with a relatively low toxic potential, comparable to water that has been treated with advanced tertiary treatments. From the present study it can be concluded that constructed wetlands may provide a complementary sewage treatment option, especially where other treatment is lacking today. To fully remove micro-pollutants from sewage effluent, however, other more advanced treatment technologies are likely needed.

Methylations in human hemoglobin

Levels of N-Methylvaline (MeVal) and N tau-methylhistidine (MeHis) were measured in male smokers and non-smokers in a program aimed at mapping background alkylations of hemoglobin (Hb) as potential indicators of doses of exogenous and endogenous genotoxic agents. MeVal was also determined in Hb from rats, Syrian golden hamsters, mice and chickens. MeVal was found to occur at levels around 0.5 nmole/g Hb, with relatively little variation between individuals and species. MeVal was not significantly affected by smoking. This result contrasts with elevated levels of N-hydroxyethylvaline (HOEtVal) measured in the same persons (Törnqvist et al., 1986b). Levels of S-methylcysteine (MeCys) (Bailey et al., 1981) and MeHis were much higher than those of MeVal. The high levels of MeCys and MeHis may be due partly to misincorporation during protein synthesis and to artifacts. S-Adenosylmethionine and formaldehyde are possible endogenous sources of MeVal. One individual (smoker) out of 21 selected for measurement of MeVal was an outlier, with raised levels of both MeVal and HOEtVal, as would be expected in case of a defective detoxification system.

Propylene oxide and epichlorohydrin induce DNA strand breaks in human diploid fibroblasts

The induction of DNA strand breaks in human diploid fibroblasts (VH‐10) was demonstrated after in vitro exposure with two carcinogenic epoxides, propylene oxide (PO) and epichlorohydrin (ECH). Alkaline DNA unwinding (ADU), pulsed field gel electrophoresis (PFGE), and the camel assay were used to measure DNA single‐ (SSBs) and double‐strand breaks (DSBs).

Role of peroxide in the radioprotective action of thiols in E. coli.

The radioprotective action of cysteamine (MEA) and cysteine in E. coli is due partly to autoxidatively generated hydrogen peroxide (H2O2). This effect, which predominates at low concentrations of the thiols (1-2 mM in neutral solution), is regularly correlated with a metabolic block, measured as inhibition of RNA synthesis. In experiments with E. coli 15 (autotroph) under exponential growth in complete medium, the role of H2O2 was demonstrated by (a) a decreased radioprotective action if catalase was present in the medium; (b) a radioprotective action of H2O2 added to the medium; (c) a decreased protective action in the absence of catalytically active copper; and (d) oxygen being required for the radioprotective action to develop. At higher concentrations of the thiols, their radioprotective action, and the accompanying metabolic block, are less dependent on H2O2 generation and presumably due to a different mechanism. The radioprotective action of H2O2 is possibly related to the radioprotective action in mammals of catalase inhibitors.

Rejoining of DNA strand breaks induced by propylene oxide and epichlorohydrin in human diploid fibroblasts

The repair kinetics of DNA single‐ and double‐strand breaks (SSBs, DSBs) induced with two carcinogenic epoxides, propylene oxide (PO) and epichlorohydrin (ECH), was studied in human diploid fibroblasts. The methods used were: alkaline DNA unwinding (ADU), the comet assay, and pulsed field gel electrophoresis (PFGE). About 70% of SSBs, measured by ADU, were rejoined after the treatment with 5 mMh and 10 mMh of PO within 20 hr, and the half‐life was estimated to be ∼15 hr. On the other hand, effective rejoining of SSBs after ECH treatment was observed only at a dose of 1 mMh (a half‐life of ∼15 hr), whereas after 2 mMh treatment, only 26% of SSBs could be rejoined within 20 hr. Furthermore, the use of the comet assay demonstrated that DNA strand breaks were effectively rejoined after PO and ECH treatment at doses of 5–10 mMh and 0.5–1 mMh, respectively. About 76% and 83% of DSBs induced by 5 and 10 mMh of PO, respectively, were rejoined within 4 hr after the treatment (a half‐life of ∼2.5 hr), with little further repair thereafter. At lower dose of ECH (1 mMh) a half‐life for DSBs rejoining was estimated to be ∼2 hr; however, only 29% of DSBs were rejoined within 2 hr at the higher dose of 2 mMh. After 18 hr, the rejoining following treatment with a lower dose was negligible. At a higher dose, a rapid accumulation of DSBs was observed, probably as the result of cell death and DNA degradation. The results demonstrate the capability of human diploid fibroblasts to repair DNA SSBs and DSBs at low‐to‐moderate doses of the epoxides. A weak capacity to rejoin DNA strand breaks induced by higher doses of ECH may be a consequence of its higher DNA alkylation activity and approximately 10 times higher toxicity compared to PO. Environ. Mol. Mutagen. 32:223–228, 1998

Lack of additive effect in mutagenesis of E. coli by UV-light and ethylene oxide

SummaryThe interaction of UV-irradiation and ethylene oxide (EtO) on forward mutation frequencies in the lacI gene of E. coli strains 3835 and 3951 and on the frequency of leu+ revertants in E. coli WU36-10-89 were studied.Pre-exposure to low doses of UV-light with the following treatment by low and intermediate doses of EtO showed lack of additive effect in the mutagenic response in all strains studied. The number of mutants actually obtained in the respective experiments was much lower than the additive model predicted.

Studies of the rad-equivalence of ethylene oxide in the presence and absence of 12-O-tetradecanoylphorbol-13-acetate (TPA) in C3H/ 10T1/2 cells

Cell transformation in vitro of C3H/10T1/2 cells, using gamma-radiation and ethylene oxide (EtO), in both the absence and presence of the cancer promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), was studied. TPA promotes transformation of C3H/10T1/2 cells to the same extent. In the dose ranges studied the average enhancement of the transformation frequency was 2.4 and 2.5 for EtO and gamma-radiation, respectively. The rad-equivalence of EtO in the presence of TPA was calculated to be 75 +/- 52 rad/mMh (95% confidence interval) which is consistent with the value 78 +/- 14 rad/mMh (95% confidence interval) obtained without TPA treatment.

Inhibition of recA induction by the radioprotector 2-mercaptoethylamine.

Our earlier finding that the radioprotective action of 2-mercaptoethylamine (MEA) is counteracted by ascorbate suggests a biochemical mechanism of action, which is supported by observations that MEA is not radioprotective in Rec- E. coli strains. In this study we show that MEA inhibits the induction of the recA gene by UV- or gamma-irradiation or by nalidixic acid in Escherichia coli strain GE94, which contains a recA-lacZ fusion. This effect, which may be counteracted by cysteine, indicates that in general MEA inhibits the induction of SOS functions.

Suppression of Induced β-galactosidase Synthesis by Cysteamine and Its Reversion by γ-irradiation in the Presence of Ascorbate

The induced synthesis of beta-galactosidase in E. coli was found to be inhibited by cysteamine. This inhibitory effect of the SH compound was antagonized by the addition of ascorbate followed by gamma-irradiation with relatively low doses. The cAMP level which, it has been suggested, plays a role in the radioprotective action of cysteamine, is stabilized by ascorbate against changes induced by irradiation.

Thiols, recA induction and radiosensitivity in Escherichia coli.

Induction by gamma-radiation, UV radiation or hydroxyurea of RecA gene product synthesis in Escherichia coli, monitored as beta-D-galactosidase in recA-lacZ fusion strains, was shown to be inhibited if 2-mercaptoethylamine (MEA) was added before treatment with the inducing agents. If cysteine (Cys) at low concentrations was added at the same time as MEA it counteracted the action of MEA. The effect of MEA may be described as a competitive inhibition of an inducing or conducting effect of Cys. In E. coli GE499 (uvrA+), complete inhibition by 30-mmol dm-3 MEA of recA induction was associated with about five times higher radio-resistence. Both of these effects of MEA were completely reversed by 0.3-mmol dm-3 Cys. As shown in parallel experiments with E. coli GE500 (uvrA-), these effects of MEA and Cys were shown to be independent of excision-repair proficiency. Treatment of bacteria with MEA and/or Cys was shown not to lead to increased intracellular concentrations of these thiols. Instead, treatment with them appeared to provoke conspicuous increases in glutathione levels, which are, however, probably not directly involved in the studied action of MEA and Cys.