Mixture and single-substance toxicity of selective serotonin reuptake inhibitors toward algae and crustaceans.

Selective serotonin reuptake inhibitors (SSRIs) are used as antidepressant medications, primarily in the treatment of clinical depression. They are among the pharmaceuticals most often prescribed in the industrialized countries. Selective serotonin reuptake inhibitors are compounds with an identical mechanism of action in mammals (inhibit reuptake of serotonin), and they have been found in different aqueous as well as biological samples collected in the environment. In the present study, we tested the toxicities of five SSRIs (citalopram, fluoxetine, fluvoxamine, paroxetine, and sertraline) as single substances and of citalopram, fluoxetine, and sertraline in binary mixtures in two standardized bioassays. Test organisms were the freshwater algae Pseudokirchneriella subcapitata and the freshwater crustacean Daphnia magna. In algae, test median effect concentrations (EC50s) ranged from 0.027 to 1.6 mg/L, and in daphnids, test EC50s ranged from 0.92 to 20 mg/L, with sertraline being one of the most toxic compounds. The test design and statistical analysis of results from mixture tests were based on isobole analysis. It was demonstrated that the mixture toxicity of the SSRIs in the two bioassays is predictable by the model of concentration addition. Therefore, in risk assessment based on chemical analysis of environmental samples, it is important to include the effect of all SSRIs that are present at low concentrations, and the model of concentration addition may be used to predict the combined effect of the mixture of SSRIs.

Ecotoxicity of mixtures of antibiotics used in aquacultures.

More or less well-defined mixtures of antibiotics used in aquacultures may be distributed in the aquatic environment. Therefore, a systematic mixture ecotoxicity study was performed with the aquaculture antibiotics oxytetracycline, oxolinic acid, erythromycin, florfenicol, and flumequine. Test organisms were freshwater algae (Pseudokirchneriella subcapitata), activated sludge microorganisms, and luminescent bacteria (Vibrio fischeri). Design and statistical analysis of test results were based on isobolographic analysis. Synergistic effects were observed when combinations of erythromycin and oxytetracycline were tested on activated sludge microorganisms, and in these cases model predictions indicate independent action on the different bacterial species in the sludge. As predicted from the modes of action, concentration addition was evident when flumequine and oxolinic acid were mixed and tested on sludge bacteria. In the algae test, the combined toxicity of antibiotics could not be predicted based on knowledge of the modes of action of the individual compounds. Independent of the test species, our results gave examples of combined effects that were higher than predicted based on the assumption of concentration addition. This result underlines the need to consider the effects of mixtures of antibiotics on environmental organisms. The isobolographic method appears to be a suitable tool for this purpose, particularly for well-defined mixtures with few substances.

Dissipation of oxytetracycline, chlortetracycline, tetracycline and doxycycline using HPLC-UV and LC/MS/MS under aquatic semi-field microcosm conditions.

A mixture of four tetracyclines; oxytetracycline (OTC), chlortetracycline (CTC), tetracycline (TC), and doxycycline (DC) was applied in fifteen 12000l outdoor microcosms at four treatment levels plus controls each with three replicates (n = 3). The dissipation times of parent compounds were monitored and half-lives (DT50) of 1-4 days, depending on treatment level were recorded. This is in accordance with half-lives from previous findings in bench-top experiments. Parent compound DT50, were determined using HPLC-UV. Furthermore, the samples were analyzed for ten different tetracycline products using LC/MS/MS. Two products were found for chlortetracycline; 4-epi-anh-chlortetracyline and the iso-chlortetracycline. Iso-forms were only found for CTC and only at the highest treatment (300 microg l(-1)). The half-lives, trajectories, and relative amounts of the products were analogous for all four tetracyclines. DT50 for products were less than 1.2 days. Formation of 4-epi-anh-tetracyclines, occurred at neutral to weak alkaline conditions.

Dissipation and effects of chlortetracycline and tylosin in two agricultural soils: a field-scale study in southern Denmark.

Presently, there is a basic lack of information concerning the accumulation of antibacterial agent residues in agricultural soils. In this field study, performed in southern Denmark, we assess the dissipation of chlortetracycline (CTC), and tylosin A (TYL A) as a function of time. Field soils were classified as a sandy loam soil (field A) and a sandy soil (field B) and each field was sampled on six occasions during the 155-d experimental period from May to October 2000 for chemical analysis and counts of colony-forming units (CFU) detecting the level of aerobic bacteria surviving antibiotic exposure. Colony-forming units and TYL A were detected throughout the entire sampling period, with respective starting soil concentrations of 30 and 50 microg kg(-1) soil declining to 1 and 5 microg kg(-1) soil, on day 155. Compound half-lives (95% confidence limits in parentheses) were estimated for both fields and T1/2 for CTC was 25 d (20-34) and 34 d (28-42) in fields A and B, respectively, and T1/2 for TYL A was 67 d (54-86) and 49 d (40-64) in fields A and B, respectively. No significant difference was determined between compound half-lives on the two fields. The level of aerobic antibiotic-resistant bacteria in the soil over time and soil fauna community was assessed in relation to application of manure containing antibacterial agents to the agricultural fields. The level of both CTC- and TYL-resistant bacteria was affected in the soil by amendment of manure, but declined during the study to the same level as observed at the beginning.

Genotoxic activity and inhibition of soil respiration by ptaquiloside, a bracken fern carcinogen.

Ptaquiloside (PTA) is a natural toxin produced by bracken (Pteridium aquilinum [L.] Kuhn). Assessment of PTA toxicity is needed because PTA deposited from bracken to soil may leach to surface and groundwater. Inhibition of soil respiration and genotoxic activity of PTA was determined by a soil microbial carbon transformation test and an umu test, respectively. In the carbon transformation test, sandy loam soil was incubated at five different initial concentrations of PTA for a period of 28 d, after which glucose was added and respiration measured for 12 consecutive hours. The tests were performed at 20 degrees C and soil moisture content of approximately 15%. For soil material sampled in the autumn, initial PTA concentrations ranging from 0.008 to 40.6 microg PTA/g dry soil were tested. From fitting of data by a sigmoidal function, a 10% effect dose (ED10) was estimated to 13 microg PTA/ g dry soil, with an upper 95% confidence limit of 43 microg PTA/g dry soil and a 95% lower confidence limit of -infinity microg PTA/g dry soil. For soil material sampled in late winter, initial PTA concentrations ranging from 1.56 to 212 microg PTA/g dry soil were tested, resulting in an ED10 value of 55 microg PTA/g dry soil, with an upper 95% confidence limit of 70 microg PTA/g dry soil and a 95% lower confidence limit of 40 microg PTA/g dry soil. The genotoxic activity of PTA was determined using the umu test without and with metabolic activation (addition of S9 rat liver homogenate). In tests with addition of S9, the induction ratio exceeded the critical ratio of 1.5 at a PTA concentration of 46 +/- 16 microg/ml and, in tests without S9, the critical ratio was exceeded at a PTA concentration of 279 +/- 22 microg/ml. The genotoxicity of PTA is comparable to that of quercetin, another bracken constituent. The toxicity of PTA toward microorganisms prolongs the persistence of PTA in terrestrial environments, increasing the risk of PTA leaching to drainage and groundwater.

Simultaneous extraction of tetracycline, macrolide and sulfonamide antibiotics from agricultural soils using pressurised liquid extraction, followed by solid-phase extraction and liquid chromatography-tandem mass spectrometry.

The veterinary antibacterial agents chlortetracycline (CTC), oxytetracycline (OTC), sulfadiazine (SDZ), erythromycin (ERY) and tylosin (TYL A, B, C and D) were extracted from soil using pressurized liquid extraction (PLE). Citric acid (pH 4.7) and methanol was used as extraction buffer, followed by tandem-solid-phase extraction (SPE) clean-up (SAX + HLB) for all compounds. For quantification two slightly different methods were employed using LC-MS-MS with MRM detection. The soil extraction method was validated using a loamy sand soil and a sandy soil, representing two typical Danish agricultural soils. Recoveries were 50-80% for the tetracyclines (CTC and OTC) and sulfadiazine (SDZ) and 60-100% for the macrolides (TYL and ERY). Limits of detection for the soil extraction method (LOD(soil)) were 0.6-5.6 microg kg(-1) soil for CTC and OTC, 0.9-2.9 microg kg(-1) soil for SDZ and 2.4-5.5 microg kg(-1) soil for TYL A and ERY. Furthermore, the method was applied to field samples taken from two agricultural fields fertilised with liquid manure containing CTC and TYL A. These results showed a decline in the content of antibacterial agents throughout the sampling period of 155 days from 10 to 15 microg CTC kg(-1) soil and 20-55 microg TYL A kg(-1) soil to below or near the LOD(soil) listed above. Finally, the method was applied to barley grains harvested from the fields. None of the antibacterial agents were measured in grain samples, but recoveries for spiked grain samples were similar to soil recoveries.

Chemical stability of chlortetracycline and chlortetracycline degradation products and epimers in soil interstitial water.

Tetracyclines and tetracycline degradation products and epimers end up in the environment. In order to predict the persistence of the potential dominating species of the chlortetracyclines in the environment, the chemical stability of chlortetracycline (CTC) and four major CTC degradation products and epimers (iso-CTC, 4-epi-CTC, anhydro-CTC, and 4-epi-anhydro-CTC) was studied in milliQ water and soil interstitial water (SIW) under environmentally relevant conditions (oxygen, light, pH (3-9), and temperature (6 degrees C and 20 degrees C)). The chemical stability of the compounds was evaluated by following the decrease in amount of parent compound over time. In order to compare the results obtained under the varying conditions, apparent pseudo-first-order rate constants (k(obs)) for the disappearance of the parent compound and corresponding apparent half-lives were calculated. A statistical evaluation of the data showed that the chemical stability of the chlortetracyclines was generally dependent on photolysis, temperature, and matrix. The presence or absence of oxygen did not influence on the chemical stability. The presence of calcium and magnesium ions in SIW is believed to account for the significant differences in half-lives between milliQ water and SIW, although numerous of other factors are believed to influence as well. Generally, the five compounds were more persistent at pH 3-4 than at pH above 5.

Evaluation of manometric respiration tests to assess the effects of veterinary antibiotics in soil.

Extensive use of antimicrobials in veterinary medicine results in environmental exposure. Of major concern are microbial effects; including effects on nutrient soil cycles and antibiotic resistance. There is a need to assess the effects of these compounds in the environment. The application of standardized guidelines is relevant in studying many compounds. However there is a lack of special test methods designed for antibiotics. We validate manometric test flasks using glucose and a recalcitrant herbicide. The suitability of these tests for studying antibacterial agents is then investigated using two target functions (aerobic biodegradation and carbon transformation). Compound stability is quantified using HPLC techniques. Effects on total soil respiration in the biodegradation test are immediate and differ significantly from background. We show that compounds do not function as substrates, so effects are due to other soil processes, correlate well to sorption characteristics and are not dose dependent. This test provides details of relative antimicrobial potency towards soil microorganisms and can be used to rank compounds. However the test does not provide details on the nature or extent of specific microbial effects. In contrast, the carbon transformation test is more specific and provides a reproducible indication of dose effect relationships, which is more suitable in assessing the effects of these compounds in the environment. Presently, standard guidelines do not take into account the normal input of antibiotics into soils via contaminated sludge or manure. This should be corrected in future guidelines as these inputs alter microbial composition, organic matter, ionic strength and pH affect sorption and overall impact the test results.

Characterisation of the abiotic degradation pathways of oxytetracyclines in soil interstitial water using LC-MS-MS.

The fate of oxytetracyclines (OTCs) in soil interstitial water was investigated and the structure of a number of degradation products elucidated in a time-related experiment. A previously developed separation method for LC-MS-MS able to base separate and quantify OTC and three of its epimers and degradation products was applied. Compounds detected were 4-epi-oxytetracycline (EOTC) (t(R)=3.0 min), OTC (t(R)=4.4 min), alpha-apo-oxytetracycline (alpha-apo-OTC) (t(R)=11.4 min) and beta-apo-oxytetracycline (beta-apo-OTC) (t(R)=18.4 min). Furthermore, we tentatively identified 4-epi-N-desmethyl-oxytetracycline (E-N-DM-OTC) (t(R)=3.0 min), N-desmethyl-oxytetracycline (N-DM-OTC) (t(R)=3.5), N-didesmethyl-oxytetracycline (N-DDM-OTC), 4-epi-N-didesmethyl-oxytetracycline (E-N-DDM-OTC) (t(R)=3.7 and 4.7 min) and 2-acetyl-2-decarboxamido-oxytetracycline (t(R)=8.7) in all samples. Most compounds were only present in trace concentrations (less than 2%) relative to the parent OTC. EOTC was on the other hand formed up to a ratio of 0.6 relative to parent OTC concentration. Only EOTC, E-N-DM-OTC, N-DM-OTC, N-DDM-OTC and E-N-DDM-OTC were formed during the time-related experiment. All other compounds were probably only present as impurities in the spiked OTC formulation as they declined in concentration from the start of the experiment. Half-lives (T(1/2), days) of the OTCs in soil interstitial water were in the order of 2 days (EOTC) to 270 days (beta-apo-OTC).

Environmental risk assessment of three selective serotonin reuptake inhibitors in the aquatic environment: a case study including a cocktail scenario.

We present an environmental risk assessment of three selective serotonin reuptake inhibitors (SSRIs; citalopram, sertraline, and fluoxetine) in the aquatic environment based on two case scenarios. Abiotic and biotic degradation experiments and sorption estimates were used to predict environmental concentrations of three SSRIs from the wastewater of two psychiatric hospitals, the primary sector, and wastewater entering and leaving wastewater treatment plants (WWTPs). Assuming a sewage treatment retention time of 8 h, abiotic degradation was low, for all three SSRIs inhibitors, ranging between 0 and 2% for hydrolysis and 0 and 6% for photolysis. The biodegradation was also slow, ranging from 0 to 3% within an 8-h period. In untreated sewage, citalopram (CIT) and sertraline (SER) concentrations may be high enough to exert effects on the aquatic biota (CIT: 0.19-10.3 µg/L; SER: 0.14-17.1 µg/L). Removal of the pharmaceuticals is due primarily to sorption in the WWTP. Sertraline was estimated to have the highest concentrations in the sewage effluents, 4.4 and 19.9 ng/L for the two cases, respectively. In treated wastewater, individual SSRI concentrations are probably too low to exert effects on biota. By using concentration addition, a cocktail exposure scenario was estimated. The predicted concentration in the biota calculated from the cocktail effect was 0.05 and 0.16 nmol/g for the two cases, respectively, and SER was found to give the highest contribution to this cocktail effect. The results indicate that the concentrations in the wastewater effluents are one to two orders of magnitude lower than the concentrations likely to cause an effect in the aquatic biota.

Leaching of estrogenic hormones from manure-treated structured soils.

The threat to the aquatic environment posed by root zone leaching of estrogens from manure-treated fields has hitherto been overlooked. The steroid hormones 17beta-estradiol (E2) and its degradation product estrone (E1) are of particular environmental concern as both are abundant in slurryfrom pregnant and cycling pigs and both are potential endocrine disruptors (lowest observable effect level (LOEL) 14 and 3.3 ng/L, respectively). The present one-year study examines the transport of E1 and E2 from manure to tile drainage systems at two field sites on structured, loamy soil. The estrogens leached from the root zone to tile drainage water in concentrations exceeding the LOEL for as long as 3 months after application, with the maximum recorded concentration of E1 and E2 being 68.1 and 2.5 ng/ L, respectively. Transport of estrogens from the soil to the aquatic environment was governed by pronounced macropore flow and consequent rapid movement of the estrogens to the tile drains. These findings suggest that the application of manure to structured soils poses a potential contamination risk to the aquatic environment with estrogen, particularly when manure is applied to areas where the majority of streamwater derives from drainage water.