Today’s urban water environment in Japan: concerns for human and ecosystem health
Figure 1 Today’s urban water environment in Japan: concerns for human and ecosystem health
Human-derived pharmaceuticals are detected in treated wastewater worldwide. In many river basins, wastewater is treated and discharged into rivers (Fig. 1). The wastewater includes human-derived pharmaceuticals and pathogens that are difficult to remove by conventional wastewater treatment processes. This raises concerns for adverse effects of pharmaceuticals on aquatic ecosystems and of pathogens to human health downstream. Our research aims at revealing the adverse effect of these contaminants on aquatic ecosystems, and at managing them on the basis of scientific evidence.
Figure 2 Neurotransmitters in the human brain
Many kinds of pharmaceuticals target the nervous system. Those that act on the G protein-coupled receptor (GPCR) include anti-hypertensives, anti-allergenics, cough medicines, and stomach medicines. Antidepressants, prescribed widely, act on the nervous system; those that target monoamine transporters include serotonin transporters (SERTs) (Fig. 2). GPCRs and SERTs occur not only in humans but also in a wide range of vertebrates and invertebrates, and therefore could have adverse effects on aquatic organisms. Many papers have reported that antidepressants can alter the behavior of fish. We are focusing on human pharmaceuticals which act on the nervous system, and investigating their effects on aquatic organisms.
Required research in environmental toxicology field
Wastewater treatment plant effluent and hospital wastewater are discharged into rivers and lakes, exposing aquatic organisms to the pharmaceuticals in them. Pharmaceuticals can induce a wide range of adverse effects from the molecular level to the population level (Fig. 3). For example, pharmaceuticals interact with target molecules in the cell (molecular-level event), changing the level of gene expression in the cell and disrupting signal pathways (cell-level event). This might alter tissue physiology (tissue- or organ-level event), leading to developmental and reproductive abnormalities, abnormal behavior, and even death (organism-level event).
Research in environmental engineering aims at detecting pharmaceuticals in wastewater and the aquatic environment, and reducing them through water and wastewater treatment. The goal of our research group is to propose how much the pharmaceuticals should be reduced by wastewater treatment. By collaborating with researchers in other fields such as molecular biology, developmental biology, and fishery science, we will propose target values for wastewater treatment on the basis of in vivo fish toxicity testing. In particular, as many pharmaceuticals to which fish are exposed affect the nervous system, we will test behavior. As at 2017, we have started in vivo testing in collaboration with fishery scientists at Nagasaki University
Figure 3 Required research in environmental toxicology field, and role of environmental engineering
Field survey of concentrations of pharmaceuticals in environmental water by in vitro assay
Figure 4 Modes of action of pharmaceuticals and in vitro assay of water samples
To determine whether pharmaceuticals in aquatic environments can alter fish behavior, we need to know the concentrations in wastewater and river water, and the concentrations that can induce abnormal behavior in fish.
To determine concentrations in water, we use an in vitro bioassay.
To determine whether the pharmaceuticals can alter behavior, we must determine the exposure and whether it is likely to elicit physiological responses, as determined by each pharmaceutical’s mode of action (MoA).
Some pharmaceuticals elicit activity by binding to receptors. For example, steroid hormones and contraceptive pills (17α-ethynylestradiol: EE2) bind to intracellular receptors, antidepressants block transporters on the cell membrane, anti-arrhythmic agents interact with ion-channels on the cell membrane, and anti-hypertensives and anti-allergenics interact with GPCRs on the cell membrane (Fig. 4). By transfecting plasmids which can express these receptors into cultured cells, exposing the cells to wastewater or river water extracts, and measuring the cell response, we can quantify the physiological activity of pharmaceuticals in aquatic environments. From the activity, the concentration can be calculated as the equivalent quantity of a representative pharmaceutical.
It is possible to measure the concentrations of selected pharmaceuticals by chemical analysis, but such concentrations do not indicate physiological activity in water. For example, even if the concentration of each substance is low, through additivity or synergism, compounds with similar MoAs might produce a strong enough physiological response to harm organisms. In contrast to chemical analysis, an in vitro assay is able to detect all compounds that interact with the receptor that is transiently expressed in the cultured cell.
Applying the in vitro TGFα shedding assay, which can measure the physiological activity of GPCR-acting pharmaceuticals, to wastewater and river water for the first time, we have detected strong activity of several types of GPCR-acting pharmaceuticals in these waters. We have also succeeded in applying an in vitro assay which can detect physiological activity induced by antidepressants such as SSRIs in waters for the first time. These techniques give us strong technical advantages in measuring the concentrations of pharmaceuticals in waters.
Understanding of the NOEC by in vivo fish exposure testing
To know the concentrations of pharmaceuticals that can induce abnormal behavior in fish, we must determine threshold concentrations by in vivo testing.
Candidate behaviors to analyze include reproduction, feeding, and avoidance of predators. In collaboration with Nagasaki University, we are exposing fish to GPCR-acting pharmaceuticals and antidepressants and monitoring their behavior by video camera. Changing the concentrations allows us to determine the no-observed-effect concentration (NOEC) of pharmaceuticals which induce abnormal behavior (Fig. 5).
Figure 5 In vivo fish exposure and behavior tests to determine no-observed-effect concentrations of pharmaceuticals
Leaving a healthy water environment for the future
Figure 6 Determining target value for pharmaceuticals during wastewater treatment
If the concentration of pharmaceuticals in wastewater measured by in vitro assay were high enough to induce abnormal behavior, such behavior could still be avoided if the concentration were reduced to below the NOEC determined by in vivo fish behavior. This approach can minimize the energy and cost required for advanced wastewater treatments such as ozonation, UV irradiation, and membrane filtration. We believe that this approach will promote compatibility between human health and aquatic ecosystems (Fig. 6).