Berndt Björlenius

Improving environmental risk assessment of human pharmaceuticals.

This paper presents 10 recommendations for improving the European Medicines Agencys guidance for environmental risk assessment of human pharmaceutical products. The recommendations are based on up-to-date, available science in combination with experiences from other chemical frameworks such as the REACH-legislation for industrial chemicals. The recommendations concern: expanding the scope of the current guideline; requirements to assess the risk for development of antibiotic resistance; jointly performed assessments; refinement of the test proposal; mixture toxicity assessments on active pharmaceutical ingredients with similar modes of action; use of all available ecotoxicity studies; mandatory reviews; increased transparency; inclusion of emission data from production; and a risk management option. We believe that implementation of our recommendations would strengthen the protection of the environment and be beneficial to society. Legislation and guidance documents need to be updated at regular intervals in order to incorporate new knowledge from the scientific community. This is particularly important for regulatory documents concerning pharmaceuticals in the environment since this is a research field that has been growing substantially in the last decades.

Comparison of six different sewage treatment processes-Reduction of estrogenic substances and effects on gene expression in exposed male fish

Treated sewage effluents often contain a mixture of estrogenic compounds in low concentrations. The total combined activity of these, however, may be sufficiently high to affect the reproduction of aquatic vertebrates. The introduction of advanced treatment technologies has been suggested as a way to remove micro-contaminants, including estrogenic substances. In this study, one municipal influent was treated with six different processes in parallel on a semi-large scale in order to assess their potential to reduce substances that could contribute to estrogenic effects in male fish. The effluent from a conventional, activated sludge treatment line was compared to a similarly treated effluent with a final sand-filtering step. The addition of ozonation (15 g O(3)/m(3)), a moving bed biofilm reactor (MBBR) or both in combination was also evaluated. There was also a separate treatment line that was based on a membrane bioreactor. A small battery of hepatic estrogen-responsive genes was measured in the exposed fish using quantitative PCR. Concentrations of steroid estrogens and estrogenic phenols in the effluents were measured by GC-ECNI-MS. The ozonated effluents were the only tested effluents for which all measured biological effects in exposed fish were removed. Chemical data suggested that the MBBR technology was equally effective in removing the analyzed estrogens; however, elevated expression of estrogen-responsive genes suggested that some estrogenic substances were still present in the effluent. The membrane bioreactor removed most of the measured estrogens and it reduced the induction of the estrogen-responsive genes. However, fish exposed to this effluent had significantly enlarged livers. Given that the same influent was treated in parallel with a broad set of technologies and that the chemical analyses were combined with an in vivo assessment of estrogenic responses, this study provides valuable input into the assessment of advanced treatment processes for removing estrogenic substances.

Reproducible 1H NMR-Based Metabolomic Responses in Fish Exposed to Different Sewage Effluents in Two Separate Studies

Treated sewage effluents contain complex mixtures of micropollutants, raising concerns about effects on aquatic organisms. The addition of advanced treatment steps has therefore been suggested. However, some of these could potentially produce effluents affecting exposed organisms by unknown modes of action. Here, (1)H NMR (proton nuclear magnetic resonance spectroscopy) metabolomics of fish blood plasma was used to explore potential responses not identified by more targeted (chemical or biological) assays. Rainbow trout was exposed in parallel to six differently treated effluents (e.g., conventional activated sludge, addition of sand filter, further addition of ozonation and/or a moving bed biofilm reactor or a separate membrane bioreactor line). Multivariate data analysis showed changes in the metabolome (HDL, LDL, VLDL and glycerol-containing lipids, cholesterol, glucose, phosphatidylcholine, glutamine, and alanine) between treatment groups. This formed the basis for postulating a hypothesis on how exposure to effluent treated by certain processes, including ozonation, would affect the metabolic profiles of exposed fish. The hypothesis withstood testing in an independent study the following year. To conclude, (1)H NMR metabolomics proved suitable for identifying physiological responses not identified by more targeted assays used in parallel studies. Whether these changes are linked to adverse effects remains to be tested.

Comparison of six sewage effluents treated with different treatment technologies--population level responses in the harpacticoid copepod Nitocra spinipes.

Since conventional treatment technologies may fail in removing many micro-pollutants, there is currently a focus on the potential of additional treatment technologies for improved sewage treatment. The aim of the present study was to evaluate six different effluents from Henriksdal Sewage Treatment Plant in Stockholm, Sweden. The effluents were; conventionally treated effluent (chemical phosphorous removal in combination with an activated sludge process, including biological nitrogen removal and a sand filter), with additional treatments individually added to the conventional treatment; active carbon filtration, ozonation at 5 mg l(-1), ozonation at 15 mg l(-1), ozonation at 5 mg l(-1)+moving bed biofilm reactor and irradiation with ultraviolet radiation+hydrogen peroxide. The evaluation was done by characterizing and comparing the effluents using a Lefkovitch matrix model based on a life cycle test with the harpacticoid copepod Nitocra spinipes, combined with analysis of juvenile development and survival over time. The conventionally treated effluent resulted in the most negative effects, leading to the conclusion that all additional treatments in the present study created effluents with less negative impacts on the copepod populations. The ozone treatments with the low dose treatment in particular, resulted in the overall least negative effects. Moving bed biofilm reactor combined with ozone did not improve the quality of the effluent in the sense that slightly more negative effects on the population abundance were seen for this treatment technology compared to ozonation alone. The active carbon treatment had more negative effects than the ozone treatments, most of which could possibly be explained by removal of essential metal ions. The effluent which was treated with ultraviolet radiation+hydrogen peroxide resulted in few developmental and survival effects over time, but still showed negative effects on the population level. Matrix population modeling proved a useful tool for biologically characterizing and comparing the effluents. Basing the assessment either on the individual level data (development and survival over time or total reproductive output) or the population level data (lambda values and projected population abundances) would not have resulted in the same conclusions as combining both analyses. The juvenile development and survival over time allowed for closer monitoring of the important molting process, whereas the population modeling provided an integrated measure of potential effects at the population level. If the dilution of the effluent in the recipient is considered, the biological effects recorded in the present study were not of substantial significance for the copepod populations, regardless of treatment technology.

Global hepatic gene expression in rainbow trout exposed to sewage effluents: A comparison of different sewage treatment technologies

Effluents from sewage treatment plants contain a mixture of micropollutants with the potential of harming aquatic organisms. Thus, addition of advanced treatment techniques to complement existing conventional methods has been proposed. Some of the advanced techniques could, however, potentially produce additional compounds affecting exposed organisms by unknown modes of action. In the present study the aim was to improve our understanding of how exposure to different sewage effluents affects fish. This was achieved by explorative microarray and quantitative PCR analyses of hepatic gene expression, as well as relative organ sizes of rainbow trout exposed to different sewage effluents (conventionally treated, granular activated carbon, ozonation (5 or 15 mg/L), 5 mg/L ozone plus a moving bed biofilm reactor, or UV-light treatment in combination with hydrogen peroxide). Exposure to the conventionally treated effluent caused a significant increase in liver and heart somatic indexes, an effect removed by all other treatments. Genes connected to xenobiotic metabolism, including cytochrome p450 1A, were differentially expressed in the fish exposed to the conventionally treated effluents, though only effluent treatment with granular activated carbon or ozone at 15 mg/L completely removed this response. The mRNA expression of heat shock protein 70 kDa was induced in all three groups exposed to ozone-treated effluents, suggesting some form of added stress in these fish. The induction of estrogen-responsive genes in the fish exposed to the conventionally treated effluent was effectively reduced by all investigated advanced treatment technologies, although the moving bed biofilm reactor was least efficient. Taken together, granular activated carbon showed the highest potential of reducing responses in fish induced by exposure to sewage effluents.

Differential removal of human pathogenic viruses from sewage by conventional and ozone treatments

Abstract Sewage contains a mixed ecosystem of diverse sets of microorganisms, including human pathogenic viruses. Little is known about how conventional as well as advanced treatments of sewage, such as ozonation, reduce the environmental spread of viruses. Analyses for viruses were therefore conducted for three weeks in influent, after conventional treatment, after additional ozonation, and after passing an open dam system at a full-scale treatment plant in Knivsta, Sweden. Viruses were concentrated by adsorption to a positively charged filter, from which they were eluted and pelleted by ultracentrifugation, with a recovery of about 10%. Ion Torrent sequencing was used to analyze influent, leading to the identification of at least 327 viral species, most of which belonged to 25 families with some having unclear classification. Real-time PCR was used to test for 21 human-related viruses in inlet, conventionally treated, and ozone-treated sewage and outlet waters. The viruses identified in influent and further analyzed were adenovirus, norovirus, sapovirus, parechovirus, hepatitis E virus, astrovirus, pecovirus, picobirnavirus, parvovirus, and gokushovirus. Conventional treatment reduced viral concentrations by one to four log10, with the exception of adenovirus and parvovirus, for which the removal was less efficient. Ozone treatment led to a further reduction by one to two log10, but less for adenovirus. This study showed that the amount of all viruses was reduced by conventional sewage treatment. Further ozonation reduced the amounts of several viruses to undetectable levels, indicating that this is a promising technique for reducing the transmission of many pathogenic human viruses.

Pharmaceutical residues are widespread in Baltic Sea coastal and offshore waters – Screening for pharmaceuticals and modelling of environmental concentrations of carbamazepine

The consumption of pharmaceuticals worldwide coupled with modest removal efficiencies of sewage treatment plants have resulted in the presence of pharmaceuticals in aquatic systems globally. In this study, we investigated the environmental concentrations of a selection of 93 pharmaceuticals in 43 locations in the Baltic Sea and Skagerrak. The Baltic Sea is vulnerable to anthropogenic activities due to a long turnover time and a sensitive ecosystem in the brackish water. Thirty-nine of 93 pharmaceuticals were detected in at least one sample, with concentrations ranging between 0.01 and 80 ng/L. One of the pharmaceuticals investigated, the anti-epileptic drug carbamazepine, was widespread in coastal and offshore seawaters (present in 37 of 43 samples). In order to predict concentrations of pharmaceuticals in the sub-basins of the Baltic Sea, a mass balance-based grey box model was set up and the persistent, widely used carbamazepine was selected as the model substance. The model was based on hydrological and meteorological sub-basin characteristics, removal data from smaller watersheds and wastewater treatment plants, and statistics relating to population, consumption and excretion rate of carbamazepine in humans. The grey box model predicted average environmental concentrations of carbamazepine in sub-basins with no significant difference from the measured concentrations, amounting to 0.57-3.2 ng/L depending on sub-basin location. In the Baltic Sea, the removal rate of carbamazepine in seawater was estimated to be 6.2 10-9 s-1 based on a calculated half-life time of 3.5 years at 10 °C, which demonstrates the long response time of the environment to measures phasing out persistent or slowly degradable substances such as carbamazepine. Sampling, analysis and grey box modelling were all valuable in describing the presence and removal of carbamazepine in the Baltic Sea.

Effects of ozonated sewage effluent on reproduction and behavioral endpoints in zebrafish (Danio rerio)

Pharmaceutical residues and other micro-contaminants may enter aquatic environments through effluent from sewage treatment plants (STPs) and could cause adverse effects in wild fish. One strategy to alleviate this situation is to improve wastewater treatment by ozonation. To test the effectiveness of full-scale wastewater effluent ozonation at a Swedish municipal STP, the added removal efficiency was measured for 105 pharmaceuticals. In addition, gene expression, reproductive and behavioral endpoints were analyzed in zebrafish (Danio rerio) exposed on-site over 21 days to ozonated or non-ozonated effluents as well as to tap water. Ozone treatment (7 g O3/m3) removed pharmaceuticals by an average efficiency of 77% in addition to the conventional treatment, leaving 11 screened pharmaceuticals above detection limits. Differences in biological responses of the exposure treatments were recorded in gene expression, reproduction and behavior. Hepatic vitellogenin gene expression was higher in male zebrafish exposed to the ozonated effluent compared to the non-ozonated effluent and tap water treatments. The reproductive success was higher in fish exposed to ozonated effluent compared to non-ozonated effluent and to tap water. The behavioral measurements showed that fish exposed to the ozonated STP effluent were less active in swimming the first minute after placed in a novel vessel. Ozonation is a capable method for removing pharmaceuticals in effluents. However, its implementation should be thoroughly evaluated for any potential biological impact. Future research is needed for uncovering the factors which produced the in vivo responses in fish.

Effect of full-scale ozonation and pilot-scale granular activated carbon on the removal of biocides, antimycotics and antibiotics in a sewage treatment plant

Several micropollutants show low removal efficiencies in conventional sewage treatment plants, and therefore enter the aquatic environment. To reduce the levels of micropollutants in sewage effluent, and thereby the effects on biota, a number of extra treatment steps are currently being evaluated. Two such techniques are ozonation and adsorption onto activated carbon. In this study, we investigated the efficiency of Swedens first full-scale ozonation treatment plant at removing a number of antibiotics, antimycotics and biocides. The effect of adding granular activated carbon (GAC) on a pilot scale and pilot-scale ozonation were also evaluated. The conventional treatment (13,000 PE) with the add-on of full-scale ozonation (0.55 g O3/g Total organic carbon (TOC)) was able to remove most of the studied compounds (>90%), except for benzotriazoles and fluconazole (<50%). Adsorption on GAC on a pilot scale showed a higher removal efficiency than ozonation (>80% for all studied compounds). Three types of GAC were evaluated and shown to have different removal efficiencies. In particular, the GAC with the smallest particle sizes exhibited the highest removal efficiency. The results demonstrate that it is important to select an appropriate type of carbon to achieve the removal goal for specific target compounds.