Vimal K. Balakrishnan

TOXICITY OF HUMAN PHARMACEUTICALS AND PERSONAL CARE PRODUCTS TO BENTHIC INVERTEBRATES

Despite concerns about potential risks associated with the presence of pharmaceuticals and personal care products (PPCPs) in the environment, few toxicological data address the effects of these compounds. In aquatic systems, which often represent the final repository for PPCPs, increasing toxicological information regarding aquatic biota is improving our capacity to assess potential risks. However, responses of key biota, such as benthic invertebrates, have not been investigated as widely. In the present study, we examined the toxicity of four PPCPs -- the lipid regulator atorvastatin (ATO), the antiepileptic drug carbamazepine (CBZ), the synthetic hormone 17alpha-ethinylestradiol (EE(2)), and the antimicrobial triclosan (TCS) -- to the midge Chironomus tentans and the freshwater amphipod Hyalella azteca in 10-d waterborne exposures. The toxicity of the four compounds varied between 0.20 and 47.3 mg/L (median lethal concentration), with a relative toxicity ranking of TCS > EE(2) > ATO > CBZ. Hyalella azteca was more sensitive than C. tentans to these compounds. The toxicity data were used in a hazard quotient approach to evaluate the risk posed by the four PPCPs to benthic invertebrates and other aquatic organisms. For each compound, a hazard quotient was calculated by dividing the lowest toxicity value by the highest exposure value found in the literature, to which an uncertainty factor was applied. With hazard quotients of 3.55 to 11.5, we conclude that potential risks exist toward benthic invertebrates for the toxicity of TCS and CBZ and that further investigations of these compounds are required to characterize more completely the risks to benthic organisms. In contrast, our data also indicate that considering the low concentrations currently detected in the environment, ATO and EE(2) pose negligible risks to benthic invertebrates.

Bioaccumulation of the synthetic hormone 17α-ethinylestradiol in the benthic invertebrates Chironomus tentans and Hyalella azteca.

The present study investigated the bioaccumulation of the synthetic hormone 17alpha-ethinylestradiol (EE2) in the benthic invertebrates Chironomus tentans and Hyalella azteca, in water-only and spiked sediment assays. Water and sediment residue analysis was performed by LC/MS-MS, while biota extracts were analyzed using both LC/MS-MS and a recombinant yeast estrogen receptor assay. At the lowest exposure concentration, C. tentans accumulated less EE2 than H. azteca in the water-only assays (p=0.0004), but due to different slopes, this difference subsided with increasing concentrations; at the exposure concentration of 1mg/L, C. tentans had a greater body burden than H. azteca (p=0.02). In spiked sediments, C. tentans had the greatest EE2 accumulation (1.2+/-0.14 vs. 0.5+/-0.05 microg/gdw, n=4). Measurements in H. azteca indicated a negligible contribution from the sediments to the uptake of EE2 in this species. These differences were likely due to differences in the behavior and life history of the two species (epibenthic vs. endobenthic). Water-only bioaccumulation factors (BAFs) calculated at the lowest exposure concentration were significantly smaller in C. tentans than in H. azteca (31 vs. 142, respectively; p<0.0001). In contrast, the sediment bioaccumulation factor (BSAF) of C. tentans was larger than that of H. azteca (0.8 vs. 0.3; p<0.0001). Extracts of the exposed animals caused a response in a recombinant yeast estrogen receptor assay, thus confirming the estrogenic activity of the samples, presumably from EE2 and its estrogenic metabolites. The results of the present study suggest that consumption of invertebrate food items could provide an additional source of exposure to estrogenic substances in vertebrate predators.

Matrix effects on mass spectrometric determinations of four pharmaceuticals and personal care products in water, sediments, and biota

Simple analytical methods were developed for the extraction and determination of four pharmaceuticals and per- sonal care products (PPCPs) from water, sediments, and biota. PPCPs were determined using tandem LC-MS in electro- spray ionization mode, and interactions with matrix co-eluents were investigated. Extractions of water samples were performed using solid-phase extraction (SPE), sediments were extracted by pressurized liquid extraction (PLE), and biota was extracted by liquid extraction. The selected analytical methods yielded recoveries ‡ 61% in all matrixes. Matrix inter- actions were investigated throughout the linear range of quantification of each compound, revealing that dissolved salts had relatively minor effects on ionization (between 14% suppression to 12% enhancement), but that sediment and biota ex- tracts caused significant matrix effects (ranging from 56% suppression to 25% enhancement). The direction and magnitude of matrix interactions reflected the physico-chemical properties of each analyte, particularly their pKa. Among the com- pounds analyzed in electrospray positive mode, carbamazepine was insensitive to matrix interactions, because it is a strong proton acceptor (pKa = 14.0). In contrast, atorvastatin (pKa = 4.5), a weaker proton acceptor, was particularly sensitive to matrix effects. For those compounds analyzed in negative-ion mode, sample alkalinity was found to be important. With a pKa of 10.4, 17a-ethinylestradiol generally exhibited matrix enhancement with increased sample alkalinity. However, the presence of acidic co-eluents contributed to matrix suppression. Lastly, TCS was particularly sensitive to matrix suppres- sion, as its circumneutral pKa (7.9) caused even slight changes in sample pH to considerably impact ionization. We con- clude that while different matrixes have clear impacts on ionization of these PPCPs, matrix effects can be quantified and overcome.

Behaviour of pharmaceuticals in spiked lake sediments – Effects and interactions with benthic invertebrates

The behaviour and effects of atorvastatin (ATO), carbamazepine (CBZ), and 17α-ethinylestradiol (EE2) were investigated in spiked lake sediments, at concentrations up to 56.5 mg kg(-1)dry weight (dw), with the benthic invertebrates Chironomus dilutus and Hyalella azteca. Desorption constants were calculated in the presence and absence of animals, using linear isotherms, yielding K(d) values of 28.2, 189.0 and 125.1 L kg(-1) (ATO), 73.7, 201.7 and 263.2 L kg(-1) (CBZ), and 114.9, 114.2 and 519.2 L kg(-1) (EE2) for C. dilutus, H. azteca, and without animals, respectively. For ATO and CBZ, K(d) values were smaller in the presence of C. dilutus, indicating greater desorption to the overlying water from bioturbation, which is consistent with the predominantly benthic occurrence of C. dilutus compared to H. azteca. In contrast, due to its greater hydrophobicity, bioturbation did not significantly affect desorption of EE2. No significant toxicity was observed, indicating decreased bioavailability of the chemicals sorbed to sediments compared with water-only toxicity assays.

Impact of selected wastewater constituents on the removal of sulfonamide antibiotics via ultrafiltration and micellar enhanced ultrafiltration

To better understand the environmental mobility of sulfonamide antibiotics and develop improved processes for their removal during wastewater treatment, stirred cell ultrafiltration (UF) experiments were conducted using both synthetic and real wastewater effluent. The interactions between selected sulfonamides (sulfaguanidine, sulfathiazole and sulfamerazine), solids and dissolved organic matter were systematically explored. The further impact of micellar enhanced ultrafiltration (MEUF), a process in which surfactants are added at micellar concentrations to enhance removal of various trace contaminants from aqueous streams, was then explored by using a cationic surfactant, cetyltrimethylammonium bromide (CTAB). Ultrafiltration of sulfonamides in the absence of other materials generally removed only 15-20% of the antibiotics. The presence of micellar solutions of CTAB generally improved removal of sulfonamides over UF alone, with rejections ranging from 20 to 74%. Environmental solids (sediment) further increased retention of sulfonamides using both UF and MEUF, but the presence of DOM did not influence rejection. Similar trends were observed on UF and MEUF of real effluent samples that had been spiked with the sulfonamides, confirming the environmental relevance of the observed interactions between sulfonamides, surfactant, and wastewater constituents. The results demonstrate that MEUF processes can be designed for the selective removal of such trace contaminants as sulfonamide antibiotics.

Chronic toxicity of the synthetic hormone 17α-Ethinylestradiol to Chironomus tentans and Hyalella azteca

The chronic toxicity of the synthetic hormone 17alpha-ethinylestradiol (EE2) was investigated in two benthic invertebrates, the midge Chironomus tentans and the freshwater amphipod Hyalella azteca, in life-cycle water-only assays. In C. tentans, a 50% decrease in emergence was observed at a concentration of 1.5 mg/L; emergence was a more sensitive endpoint than survival, growth, or biomass. Reproduction was not significantly affected by EE2 exposure until a concentration of 3.1 mg/L, where emergence, and therefore reproduction, did not occur. In contrast, reproduction was the most sensitive endpoint in H. azteca (50% decrease in reproduction observed at a concentration of 0.36 mg/L). The sensitivity of the F1 generation to EE2 was also investigated with H. azteca, but was not different from the F0 generation. The data from the present study were combined with those from previous 10-d toxicity assays, to derive acute to chronic toxicity ratios (ACRs) for EE2. The ACRs calculated for EE2 were 13 for C. tentans and 16 for H. azteca, indicating that the application factors currently used in ecological risk assessment for the derivation of chronic toxicity are protective and conservative for these organisms. The results of the present study suggest that chronic toxicity was not mediated by disruption of endocrine pathways. Using a hazard quotient approach, the risk associated with sublethal exposure to EE2 was <<1 for H. azteca and C. tentans, indicating that adverse effects are not expected, and that environmental exposure to EE2 likely poses a low risk to benthic invertebrates.

Anthropogenic impacts on a bedrock aquifer at the village scale.

This study focuses on assessing groundwater potability in a highly complex and heterogeneous fractured bedrock aquifer having variable overburden cover. Eight monitoring wells were installed in a privately serviced lakeside village, and groundwater was routinely sampled over a 2-year timeframe for concentration analysis of nitrate, fecal indicator bacteria, stable isotopes, and a total of 41 pharmaceutical compounds. While pollutant concentrations remained low throughout the study, the presence of fecal indicator bacteria and pharmaceuticals was noted at least once (but not always consistently) in most sampling intervals. An interpretation based on the integration of chemical, bacterial, and site characterization datasets suggests that: (1) the fracture network is complex and heterogeneous with limited vertical connectivity; (2) existing pathways are sufficient for the quick and widespread migration of surface contaminants to depth; (3) anthropogenic contaminants from both septic systems and agriculture are likely sourced in the surrounding uplands where overburden is thin; and (4) fecal contamination, as observed over the long term, is ubiquitous at the village scale. Groundwater quality is continually changing in this hydrogeologic environment and the determination of potability on the larger scale is not likely to be adequately captured with infrequent domestic well sampling (i.e., voluntary annual sampling by homeowners).

Life‐cycle exposure of fathead minnows to environmentally relevant concentrations of the β‐blocker drug propranolol

Propranolol is a human pharmaceutical β-blocker that has been detected in municipal wastewater effluents at ng/L to low μg/L. To assess the potential of this compound to affect fish, fathead minnow (Pimephales promelas) were exposed for a life cycle in a flow-through system to nominal propranolol concentrations of 0.87 ng/L, 8.7 ng/L, 87 ng/L, 870 ng/L, and 8700 ng/L. Measured propranolol concentrations were below detection for the 2 lowest exposure concentrations, and were 76 ng/L, 580 ng/L, and 7800 ng/L for the 3 highest exposure concentrations. During the 162-d to 165-d exposure, no significant changes in weights or lengths were seen in fathead minnows, although the highest concentration of propranolol did cause a 15% decrease in survival of larval and juvenile stage fish compared with controls. At maturity, there were no significant changes in condition factor, liver-somatic index, or secondary sex characteristics in propranolol-exposed male or female fish. Female gonadosomatic index was significantly decreased in fish exposed to the highest concentrations of propranolol, probably because of increased egg-laying. Fathead minnows from all propranolol exposures produced more eggs than control fish, with fish exposed to 7800 ng/L propranolol producing 70% more eggs per female (p = 0.060), and having significantly increased clutch size (p = 0.008). Egg quality, % fertilization, % hatching, and % deformities in F1 fry were unaffected by propranolol exposure of fish. Propranolol exposure caused no effects in fathead minnows, except at the highest exposure concentration (7800 ng/L), where there were slight decreases in survival of juvenile minnows, and indications of increased reproduction. The present study is important because it is the first to assess the potential for effects in fish exposed to propranolol for a life cycle. Environ Toxicol Chem 2017;36:1644-1651.