Manifestation of Ecotoxicity in Parts per Trillion Contaminant Levels in

Contaminants of Emerging Concern in the Environment: Ecological and Human Health Considerations Downloaded from pubs.acs.org by STONY BROOK UNIV SUNY on 01/31/18. For personal use only.

Chapter 16

Manifestation of Ecotoxicity in Parts per Trillion Contaminant Levels in Natural and Simulated Environmental Settings Hans Sanderson* and Martin Rudbeck Jepsen University of Aarhus, National Environmental Research Institute, Department Policy Analysis, 399 Frederiksborgvej, 4000 Roskilde, Denmark *E-mail: [email protected]; Tel. +45 4630 1822.

Over the past decades a blend of different active pharmaceutical ingredients (APIs) have been detected in environmental settings at trace amounts across the globe. The continual release of APIs, primarily into the aquatic environment, has earned the APIs a status as ubiquitous and pseudo-persistent contaminants of emerging concern. The question is however, if parts per trillion levels of APIs may manifest measureable toxicities and risks to the environment? The acute risks are typically neglible low from these low exposures, but the chronic risks may potentially be overlooked. This paper demonstrates three case-studies where unexpected and long-term effects were observed for three different APIs. This illustrates that chronic toxicity may occur in some cases even at very low concentrations. To detect the manifestation of toxicity at parts per trillion levels more effectively will require more specific and tailored tests relevant to the individual drug and organism of concern. There are currently being developed novel methods and frameworks that will allow a prioritization of APIs of specific concern for further tailored testing, which are briefly presented herein.

1. Introduction Contaminants of emerging concern challenge environmental toxicology as we know it (1). We are able to detect manmade compounds in the environment at ever lower concentrations and at increasing speed; therefore, the list of emerging © 2010 American Chemical Society


environmental contaminants in water and other compartments is growing (2). Detection of trace amounts of individual compounds and mixtures raises the question of whether they may cause toxicity or even present an appreciable risk to humans and the environment? Both the awareness of the presence of the compounds in the environment are emerging, as well as concern surrounding the impacts of these compounds. The latter is uncertain and questions arise with regard to what the hazards are, whether risk is present at low concentrations, and whether this risk can be detected with current techniques and methods? This chapter will review three case studies from natural and semi-natural environmental settings and demonstrate how pharmaceuticals among contaminants of emerging concern bring about impacts, and even potential risks to the environment at very low concentrations. The first case study examines how trace amounts of the non-steroidal anti-inflammatory drug, diclofenac resulted in significant reductions in the Pakistani population of oriental white-backed vulture (Gyps bengalensis), as reported by Oaks et al. in 2004 (3). The second case study presents experiments undertaken by Kidd et al. (4) with the synthetic estrogen 17α-ethynylestradiol (EE2), used in birth-control pills. Kidd et al. (4) treated a Canadian lake with traces of EE2 comparable to the levels found downstream of municipal wastewater treatment plants, and found a collapse of the fathead minnow (Pimephales promelas) population in the lake. The third and last case study is a mesocosm test under semi-field conditions with the antiparasitic drug, ivermectin undertaken by Sanderson et al. (5). During this study, the fate and effects of the drug was monitored for one year in 12,000 litre aquatic mesocosms, with focus on the partitioning of the drug between water and sediment as well as on the ecosystem effects. Finally, we will reflect upon research needs for improved testing of APIs.

2. Case Studies The purpose of the three case studies is to illustrate how trace amounts of active pharmaceutical ingredients manifested ecotoxicity and risks towards nontarget organisms. There are several studies that demonstrate ecotoxicity at low levels (3–6). However, the three studies presented here also illustrate risks at these levels and cover widely used drugs, two of which are used both for veterinary and human purposes (diclofenac and ivermectin), and the remaining drug is a hormone (EE2) widely used in the human population. The case studies are large scale and long term, with focus ranging from very large ecosystems in Pakistan to experimental lakes facility in Ontario, Canada and an experimental mesocosm study facility. In the latter the ecosystem was replicated, allowing more robust statistical evaluation of the observations, as does the experimental lake facility in Ontario. 2.1. Diclofenac and Pakistani Vultures Diclofenac is a non-steriodal anti-inflamatory drug (NSAID) used to reduce pain and inflammation in humans and animals. It was introduced as a treatment 338


for livestock on the Indian subcontinent in the early 1990s, coinciding with the beginning of a rapid decline in Gyps vulture populations. It took approximately 10 years to demonstrate that this was more than a coincidence. In 2004, Oaks et al. published a study in Nature (3) in which very clear evidence that diclofenac is lethal to Gyps vultures was presented. In their study undertaken in Pakistan in 2000-2003, the authors examined 219 dead oriental white-backed vultures (OWBVs), of which 85% showed characteristics of visceral gout in their internal organs (as evidenced by subsequent deposition of uric acid on and in their organs). Of the 219 OWBVs, 42 were found within 24 hours after death and detailed necropsies were performed. 14 of these showed no signs of visceral gout (other causes of death could be established in eight cases), while the remaining 28 with visceral gout displayed severe, acute renal tubular necrosis. Among the 28 cases, only one had an identifiable infection and no toxic concentrations of heavy metals, pesticides or pathogens were detected. For 25 of the 28, liver samples were tested for diclofenac residues. Residuals were found in all samples at concentrations ranging from 0.051 to 0.643 μg g-1 (unadjusted wet weight (w.w.)). Of the 14 OWBVs without visceral gout, samples were taken from 13, and none of them showed diclofenac residues. The next step by Oaks et al. (3) was to administer veterinary diclofenac orally to 4 captive vultures. The recommended mammalian dose of 2.5 mg kg1 was given to two vultures while the remaining other two received a smaller dose of 0.25 mg kg-1. All four birds developed hyperuricaemia within 24 hours, and three of the four birds died within 36-58 hours after administration. Finally, 10 juvenile OWBVs were fed meat from cattle or goats that had received the recommended therapeutic dose of diclofenac intramuscularly once daily for three days and were slaughtered 4 hours after the last injection. The resulting diclofenac residues in meat and organs of these livestock ranged from 0.19 to 5.7 μg g-1 (w.w.). Additionally, 10 OWBVs were fed meat containing 6.4 μg g-1 of diclofenac. By accounting for the meat consumed by the birds, eight OWBVs received doses between 0.005 and 0.3 mg g-1, resulting in death by renal failure in two of these. Two OWBVs received doses of 0.5 to 0.6 mg g-1, one of them died of renal failure. Ten birds consumed meat corresponding to doses of 0.8 to 1.0 mg g-1, all of which died from renal failure. The resulting mortality rate of 13 deaths among 20 vultures (65%) demonstrates a significant relationship between exposure to diclofenac and renal failure. It is now accepted that scavenging on livestock carcasses treated with diclofenac shortly before death posses a serious threat to Gyps vultures, and contamination with diclofenac of just one out of 760 livestock carcasses is sufficient to explain the collapse in Gyps vulture populations on the Indian subcontinent (7). The 80% to 95% collapse of the OWBV population lead to a rise in the number of scavenging stray dogs, which in turn resulted in an increase in rabies incidents in humans due to contact with these dogs. As a result, on 11 May 2006, the Drug Controller General of India ordered the withdrawal of all licenses granted for the manufacture and veterinary use of diclofenac in India (8).

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2.2. EE2 and the Fathead Minnows in a Canadian Lake It is well known that the presence of estrogenic substances is associated with reproductive endocrine disruption in male fish populations (9), leading to vitellogenin (VTG) production and early-stage egg development in their testes. However, other synthetic estrogen mimicking compounds and pathways have been suggested as other causative agents (10). Estrogens have been found in surface waters and wastewater effluents at concentrations of 17 and up to 147 ng l-1, measured as equivalents of the natural estrogen 17β-estradiol (E2) (11). In a recent whole-lake study conducted from 1999 to 2005 in Ontario’s Experimental Lakes Area, Kidd et al. (4) describes the effect of constantly exposing a population of fathead minnow (Pimephales promelas) to the synthetic estrogen 17α-ethynylestradiol (EE2) for three consecutive years (2001-2003). The fathead minnow has a lifespan of up to 4 years and reaches sexual maturity during the second year of life. EE2 was added at concentrations similar to, or below those found in rivers receiving water from wastewater treatment plants, with seasonal mean values in the experimental lake kept from 4.8 to 6.1 ng l-1. The fathead minnow population was regularly sampled, and gonadal development as well as VTG mRNA and protein concentrations in the exposed fish were compared to populations of fathead minnow in two non-exposed experimental lakes. Seven weeks after the experiment started, whole-body concentrations of VTG in males were three orders of magnitude greater than reference samples, and normalized liver VTG mRNA values in males were approximately a factor 100 greater than values found in males in reference lakes and more than an order of magnitude greater than the levels found in female fish in the reference lakes. In the spring of 2002, less than a year after the EE2 additions began, testicular tissues from sampled males showed delayed spermatogenesis, widespread fibrosis, and malformations of the tubules. The following year, 2003, almost half (~45%) of the males captured had ova-testes displaying primary-stage oocytes. The impact of EE2 additions was striking. In the second year of exposure, no young-of-the-year were caught, and the adult population had severely declined compared to abundance pre-exposure and in the reference lake. After the three years of EE2 additions the fathead minnow population had completely collapsed (4). 2.3. Ivermectin and the Pelagic Ecosystem Structure and Function Antiparasitic drugs are among the most important groups of veterinary pharmaceuticals in the European Union (EU), with a market volume of more than 200 million euro. Ivermectin (CAS# 70288-86-7) is one of the most widely used macrocyclic lactones and has been known as a potent, effective and safe antiparasitic drug since 1981. The compound inhibits the signal transmission at GABA-gated and glutamate-gated chloride channels in the target parasite causing death. GABA receptors respond to the neurotransmitter gamma-aminobutyric acid (GABA), which is the the major inhibitory neurotransmitter in the vertebrate central nervous system (CNS). Ivermectin shots down the CNS in the target organism thus causing death. The compound is commonly used for treatment and 340


prevention of internal and external parasites of cattle, horses, and other animals on pasture (e.g. gastrointestinal and respiratory tract nematodes, flies, grubs, ticks, lice, and mites) (5). Environmental assessment of ivermectin found that Daphnia magna was the most sensitive species with a 48 hr leathal concentration for 50% of the test organisms (LC50) = 25 ng L-1, and a no observed effect level (NOEL) at ~ 10 ng L-1. Due to a high organic carbon adjusted sorption coefficient (Koc > 12.600), and rapid dissipation in water, with a half-life (DT50) of between 12 and 39 hrs, the calculated and measured worst-case environmental concentrations in surface waters ranged from 2 to 25 ng l-1. The investigation described in this case study commenced in August 2004 at the University of Guelph Mesocosm Facility in Ontario, Canada (latitude 43.5 ° N), and ended in May 2005. The facility consists of 30 mesocosms that are sunk into the ground and designed to replicate natural pond systems. The mesocosms are approximately 1.2 m deep with a water depth of 1 m, a diameter of 3.9 m and a surface area of 11.95 m2, with a volume of approximately 12,000 l of water. The treatment regime was in triplicate (n = 3): Controls; 30; 100; 300; and 1,000 ng l-1 (parts per trillion (ppt)) nominal concentrations). Water and sediment concentrations were monitored as well as the biotic and abiotic parameters of interest. All the measured ivermectin concentrations at the beginning of the study, after treatment, were between 10 and 25% below the nominal concentrations. The aquatic DT50 was determined to be 4 days. Ivermectin built up over the first 3-4 weeks following treatment and then stabilized at between 20 and 30 μg kg-1 for the highest treatment levels. Cladoceran and Copepod species richness, Ephermeoptera abundance, DO and pH were significantly impacted during the chronic phase (day 10 to 100), where the measured ivermectin water concentration was below the detection limit (1 ng l-1), and approximately 25 ng kg-1 in the sediment, suggesting potentially severe chronic risk (aquatic risk quotient = 25) to the zooplankton communities as well as more indirect ecosystem functional parameters such as pH and dissolved oxygen. The long-term effects (day 100 to 265) remained evident only for Ephermeoptera abundance and the cladoceran Chydorous spp after the winter and into the spring, and at this time ivermectin was no longer detectable in the water phase, only in the sediment. Ephermeoptera and Chydorous spp. are relatively more active in the sediment compartment than the majority of the other species monitored (12) and a relative, increasing, long-term sensitivity is thus assumed due to relatively elevated exposure via the sediment. Ephermeoptera and Chydorous spp. had not recovered by the end of the study, almost one year after treatment (5).

3. Discussion and Conclusions On the basis of these three case studies, we can conclude that ecotoxicity can manifest itself at parts per trillion levels. Some effects are unexpected, such as the collapse of the vulture population in Pakistan, some are potentially severe at realistically low levels as demonstrated by EE2 and the fathead minnow collapse, and some may show long-term structural and functional effects in aquatic ecosystems (ivermectin). Impacts have received the attention of regulators, as 341


we heard from India with regards to diclofenac, but also targeted removal of endocrine disrupting compounds (hereunder EE2) from municipal wastewater has come under consideration (13). Continued veterinary use of ivermectin has also been scrutinized, both with regards to its impact on dung beetles and thus pasture productivity, but also with regard to its use in aquaculture to treat sea lice in salmon (see e.g. the FP6 project ERAPharma where ivermectin was the subject of one of three case studies http://www.erapharm.org/index.html). How can ecotoxicology in future address similar surprises and unintended impacts on the environment as well as help avoid them? Can we adapt our testing regimes to elucidate these subtle effects? As previously mentioned, pharmaceuticals in trace amounts present a challenge for ecotoxicology, but there are actually a number of tools available that can guide future chronic impact assessments. One of these tools is the Comparative Toxicogenomics Database (CTD) (http://ctd.mdibl.org/). Here it is possible to identify the gene interactions of individual compounds as well as subsequent proteins and signalling pathways. For diclofenac interaction is with CYP2CP, and we can see that diclofenac shares 19 common gene interactions with another NSAID naproxen, suggesting that naproxen might not be a suitable substitute. Meloxicam would probably be a better substitute from a Gyps vulture protection persepective as it does not share gene interactions with diclofenac (14). Ivermectin interacts with the ABCB1 (ABC being the ATP Binding Cassettes), a widespread protein family in many species and levels of biological organization, and ivermectin may cause liver cirrhosis in humans. EE2 is well known to interact along the hypothalamic-pituitary-gonadal (HPG) axis. Furthermore, the ToxCast program (http://www.epa.gov/ncct/toxcast/), hereunder the Aggregated Computational Toxicology Resource (ACToR) (http://actor.epa.gov/actor/faces/ACToRHome.jsp) is a user friendly resource that rapidly can provide an overview of a compound’s toxicity. Armed with screening analyses using these and other similar databases and tools (15), it is possible to address and investigate concerns about individual active pharmaceutical ingredients as well as other chemicals more specifically, and to suggest more specific toxicity assessments for each compound, thereby avoiding a one-size-fits-all testing system. Huggett et al. (2003) (16) suggested a screening approach based on a comparison between the human therapeutic plasma concentration of a drug to the measured or calculated steady-state plasma concentration in fish, and thereby derive a relative risk ranking. Ankley et al. (2007) (17), suggest screening and prioritization of APIs for further investigation based on assessment of the exposure potential based on production volume and predicted environmental concentrations. The next tier would be assessment of the presence target pathways that may lead to toxicologically relevant responses, i.e. an assessment of the Modes of Action. This approach has recently been further developed, in a systems biological framework designed to elucidate the adverse outcome pathway (AOP), which portrays the linkage between molecular events and an adverse outcome at a biological level relevant to risk assessment (18). With these analysis and prioritization it would be possible to determine relevant assessment endpoints tailored for the germane specific questions concerning the manifestation of APIs chronic risks towards ecosystems at parts per trillion levels. 342

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Manifestation of Ecotoxicity in Parts per Trillion Contaminant Levels in

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