Dialysis experiments for assessing the pH-dependent sorption of sulfonamides to soil clay fractions.

Equilibrium dialysis experiments, a novel approach for conducting soil/water distribution experiments in environmental samples, were found to be applicable for assessing pH-dependent partitioning and to quantify the sorption of three sulfonamides, sulfadiazine, sulfadoxine, and sulfacetamide. Clay fractions from two agricultural soils including both particulate and dissolved soil matter were used in the experiments to achieve a high sorption capacity when varying pH in a relevant environmental range. Stabilizing and controlling pH was done by using organic buffers. In two clay fractions, Kd for sulfadiazine was determined to be 43 and 129 L kg(-1), and 1.3 and 4.6 L kg(-1) at pH 4.0 and pH 9.0, respectively. This corresponded to Kd for the neutral and ionized form of sulfadiazine, respectively. The difference in sulfadiazine sorption between the two clay fractions could to some extent be related to the difference in the amount of organic carbon. Sorption experiments with sulfacetamide and sulfadoxine also exhibited decreasing sorption when increasing pH. At low pH, maximum Kd for sulfacetamide and sulfadoxine was determined to be 83 and 211 L kg(-1), respectively, while at high pH minimum Kd was 4.8 and 1.2 L kg(-1), respectively. Hence, compound speciation was important for the quantity of sorbed sulfonamide, which was confirmed by a correlation (R(2)) close to unity, when using the experimentally obtained Kd values with a simple model weighing the contribution from the neutral and the ionized compound, respectively.

Suggesting a testing strategy for possible endocrine effects of drug metabolites.

Most pharmaceuticals are extensively metabolized by organisms, which results in internal exposure to mixtures of parent compounds and various metabolites. Many of these metabolites are considered non-toxic, but some metabolites retain toxic properties of the parent compound or elicit other undesirable outcomes. Unfortunately, the effects of metabolites are often not considered when endocrine activities of chemicals are evaluated in vitro. In this study two approaches, an "effect-based" and a "compound-by-compound" testing design, were used to determine the effects of metabolites of the antidepressant sertraline on aromatase enzyme activity. In the "effect-based" approach, a mixture of sertraline metabolites, produced by liver microsomes, inhibited aromatase, but was less potent than sertraline. In the "compound-by-compound" testing design, three specific metabolites were evaluated individually and in mixtures. Though two N-desmethylated metabolites were more potent aromatase inhibitors than sertraline, hydroxyl ketone sertraline did not inhibit the enzyme and mixtures of these metabolites and sertraline were less potent than predicted from a concentration addition model. Our findings highlight the importance of considering aromatase inhibition, and potentially other biological activities, of pharmaceutical metabolites produced by liver microsome preparations and then comparing such observations to studies of specific metabolites available for testing in pure form. Subsequently, a five step integrated strategy for screening of the potential endocrine effects of drugs and their metabolites are proposed.

Sorption and mobility of ivermectin in different soils.

Avermectins are widely used to treat livestock for parasite infections. Ivermectin, which belongs to the group of avermectins, is particularly hazardous to the environment, especially to crustaceans and to soil-dwelling organisms. Sorption is one of the key factors controlling transport and bioavailability. Therefore, batch studies have been conducted to characterize the sorption and desorption behavior of ivermectin in three European soils (Madrid, York, and artificial soil). The solid-water distribution coefficient (K(d)) for ivermectin sorption to the tested soils were between 57 and 396 L kg(-1) (determined at 0.1 microg g(-1)), while the organic carbon-normalized sorption coefficients (K(oc)) ranged from 4.00 x 10(3) to 2.58 x 10(4) L kg(-1). The Freundlich sorption coefficient (K(F)) was 396 (after 48 h) for the artificial soil over a concentration range of 0.1 to 50 microg g(-1), with regression constants indicating a concentration-dependent sorption. The obtained data and data in the literature are inconclusive with regard to whether hydrophobic partitioning or more specific interactions are involved in sorption of avermectins. For abamectin, hydrophobic partitioning seems to be one of the dominant types of binding, while hydrophobicity is less important for ivermectin, which is probably due to the lower lipophilicity of the molecule. Furthermore, the presence of cations such as Ca(2+) leads to decreasing sorption. Thus, it is presumed that ivermectin binds to soil by formation of complexes with immobile, inorganic soil matter. In contrast to abamectin, hysteresis could be excluded for ivermectin in the studied soils for the evaluation of sorption and desorption. The sorption mechanism is highly dependent on physicochemical properties of the avermectin.

Development of an analytical method to determine avermectins in water, sediments and soils using liquid chromatography-tandem mass spectrometry.

A comprehensive analytical multi-residue method has been developed for the determination of seven avermectins (abamectin, doramectin, ivermectin, emamectin benzoate, eprinomectin, moxidectin and selamectin) in surface water, sediment and soil samples. Solid samples were extracted applying pressurised liquid extraction followed by a solid-phase extraction (SPE) clean-up step. For aqueous samples, extraction was done utilising only SPE. All compounds were measured using liquid chromatography coupled to tandem mass spectrometry using atmospheric pressure chemical ionisation. The recoveries were 38-67% (relative standard deviation: 9-26%), 63-88% (16-23%) and 63-80% (9-15%) for spiked Rhine water, spiked sediment and spiked soil samples, respectively, and limit of quantifications were 2.5-14 ngl(-1) in water and 0.5-2.5 ngg(-1) in soil and sediment.

Determination of ivermectin and transformation products in environmental waters using hollow fibre-supported liquid membrane extraction and liquid chromatography-mass spectrometry/mass spectrometry.

A simple and easy-to-use extraction method for aqueous samples based on hollow fibre-supported liquid membrane (HF-SLM) followed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) was developed to determine ivermectin and transformation products, the monosaccharide (TP1) and the aglycon of ivermectin (TP2). The proposed method attained enrichment factors up to 80, after optimising parameters, such as fibre length, organic solvent, stirring speed, salt level, pH in samples/fibre, extraction time and fibre emptying technique. Method validation with tap and lake water samples provided good linearity and detection limits of 0.2, 1.6 and 0.9 microg/l for ivermectin, TP1 and TP2 in lake water with RSD below 15%.

Inhibition of human aromatase complex (CYP19) by antiepileptic drugs.

Antiepileptic drugs and epilepsy are often associated with sexual disorder in women such as hyperandrogenism, menstrual disorders and ovarian cysts. In children, until puberty, a hormone imbalance may influence many aspects of development, e.g. growth and sexual maturation. The aromatase complex is the enzyme system that converts androgens to estrogens and consequently an inhibition may induce a hormone imbalance. Twelve antiepileptic drugs, used in mono or polytherapy for the treatment of children, were tested for their ability to inhibit aromatase (CYP19) with commercially available microsomes from transfected insect cells using dibenzylfluorescein as substrate. The drugs inhibiting CYP19 were: lamotrigine, oxcarbazepine, tiagabine, phenobarbital, phenytoin, ethosuximide, and valproate. The inhibitory effects (50% reduction in activity compared to enzymes without inhibitor present) were in the range of 1.4-49.7 mM. Carbamazepine, gabapentin, primidone, topiramate and vigabatrin showed no inhibition. Additionally, binary drug combinations were tested to investigate if combination therapy could potentiate the aromatase inhibition. Additive inhibition was seen in combination experiments with valproate and phenobarbital. When adding carbamazepine to a range of valproate concentrations no additional inhibition was seen. The data for some of the AEDs show that side effects on steroid synthesis in humans due to inhibition of aromatase should be considered.

Reduced antimicrobial potencies of oxytetracycline, tylosin, sulfadiazin, streptomycin, ciprofloxacin, and olaquindox due to environmental processes.

The stability of oxytetracycline (OTC), tylosin (TYL), sulfadiazin (SDZ), streptomycin (ST), ciprofloxacin (CF) and olaquindox (O) was examined in environmentally relevant matrices, such as soil interstitial water and sewage sludge water. Compounds were assessed in both aerobic (OTC, TYL, SDZ, ST, and CF) and anaerobic experiments (OTC, TYL, and O) using analytical measurements (UV spectrophotometry or HPLC) combined with a growth inhibition pour plate assay using activated sludge bacteria. (OTC was additionally assessed using a soil bacterial assay.) This combination of results enabled the assessment of whether a loss in antibacterial potency was reflected in a similar reduction of substance concentration. If a potency reduction is not reflected in a decreased substance concentration, the results may indicate the formation of less potent degradation products possessing the same chromophoric system (same UV absorbance maximum) as the parent compound. With the exception of ST and CF, the antimicrobial agents generally lost a considerable amount of their antimicrobial potency in aerobic experiments. In the anaerobic experiments having either an experimental duration of 21 or 100 days only OTC retained potency. These results correspond well with the fact that several degradation products were encountered in the study for this compound

Environmental properties and effects of nonionic surfactant adjuvants in pesticides: a review.

Little is known about the environmental fate of adjuvants after application on the agricultural land. Adjuvants constitute a broad range of substances, of which solvents and surfactants are the major types. Nonionic surfactants such as alcohol ethoxylates (AEOs) and alkylamine ethoxylates (ANEOs) are typically examples of pesticide adjuvants. In view of their chemical structure this paper outlines present knowledge on occurrence, fate and effect on the aquatic and terrestrial environment of the two adjuvants: AEOs and ANEOs. Both AEOs and ANEOs are used as technical mixtures. This implies that they are not one single compound but a whole range of compounds present in different ratios. Structurally both groups of substances have a mutual core with side chains of varying lengths. Each of these compounds besides having the overall ability to distribute between different phases also possesses some single compound behaviour. This is reflected in the parameters describing the fate e.g. distribution coefficient, leaching, run-off, adsorption to soil, degradation and effects of these substances. The adsorption behaviour of ANEOs in contrast to AEOs is particularly variable and matrix dependent due to the ability of the compound to ionise at environmentally relevant pH. Probably because the compounds exceeds high soil adsorption and are easily degradable which is reflected in the low environmental concentrations generally found in monitoring studies. The compounds generally possess low potency to both terrestrial and aquatic organisms. The major environmental problem related to these compounds is the ability to enhance the mobility of other pollutants in the soil column.

Toxicity of tetracyclines and tetracycline degradation products to environmentally relevant bacteria, including selected tetracycline-resistant bacteria.

Tetracyclines used in veterinary therapy invariably will find their way as parent compound and degradation products to the agricultural field. Major degradation products formed due to the limited stability of parent tetracyclines (tetracycline, chlortetracycline, and oxytetracycline) in aqueous solution were theoretically identified at various environmental conditions, such as pH, presence of chelating metals, and light. Their potency was assessed on sludge bacteria, tetracycline-sensitive soil bacteria, and tetracycline-resistant strains. Several of the degradation products had potency at the same concentration level as tetracycline, chlortetracycline, and oxytetracycline on both the sludge and the tetracycline-sensitive soil bacteria. Further, both 5a,6-anhydrotetracycline and 5a,6-anhydrochlortetracycline had potency on tetracycline-resistant bacteria supporting a mode of action different from that of the parent compounds.

Inhibition of aerobic growth and nitrification of bacteria in sewage sludge by antibacterial agents.

Toxicity of antibacterial agents on environmentally relevant bacteria was investigated using activated sludge. The growth and nitrifying inhibiting effects for activated sludge of benzyl penicillin (penicillin G) (BP), tetracycline (TC), chlortetracycline (CTC), oxytetracycline (OTC), olaquindox (O), streptomycin (ST), tiamulin (TI), tylosin (TYL) sulfadiazine (SDZ), metronidazole (MET), and oxolinic acid (OXA) was investigated. Studies were performed in accordance to the ISO 15522 (1999) and ISO 9509 (1989) test guidelines, respectively. The toxicity (EC50 value, mg/L) found with the ISO 15522 was in decreasing EC50 values; O (95.7), BP (84.6), TYL (54.7), TI (14.3), TC (2.2), OTC (1.2), ST (0.47), CTC (0.40), and OXA (0.1). No observed effect concentrations (NOECs) (mg/L) of 100 and 60, respectively was found for MET and SDZ. Triplicate tests assessing the effects of the antibacterial agents on the nitrification rate gave indications only as the level of increased or decreased rate. More accurate data for the inhibition of Nitrosomonas europaea was found with a suspended culture of the nitrifying bacteria. The toxicity (EC50 value, mg/L) found was in decreasing EC50 values; TI (23.3), SDZ (17.0), TC (4.0), OTC (1.7), OXA (1.0), CTC (0.64), O (0.03), ST (0.02). For MET and TYL, NOECs (mg/L) of 100 and 50 were found, respectively. The antibacterial agents were also assessed using a pour plate method with both (separately tested) activated sludge bacteria and N. europeae showing to be 5 to 10 times more potent to most agents except SDZ, TI, and MET.

Primary biodegradation of veterinary antibiotics in aerobic and anaerobic surface water simulation systems.

The primary aerobic and anaerobic biodegradability at intermediate concentrations (50-5000 microg/l) of the antibiotics olaquindox (OLA), metronidazole (MET), tylosin (TYL) and oxytetracycline (OTC) was studied in a simple shake flask system simulating the conditions in surface waters. The purpose of the study was to provide rate data for primary biodegradation in the scenario where antibiotics pollute surface waters as a result of run-off from arable land. The source of antibiotics may be application of manure as fertilizer or excreta of grazing animals. Assuming first-order degradation kinetics, ranges of half-lives for aerobic degradation of the four antibiotics studied were 4-8 days (OLA), 9.5-40 days (TYL), 14-104 days (MET) and 42-46 days (OTC). OLA and OTC were degraded with no initial lag phase whereas lag phases from 2 to 34 days (MET) and 31 to 40 days (TYL) were observed for other substances. The biodegradation behaviour was influenced by neither the concentrations of antibiotics nor the time of the year and location for sampling of surface water. Addition of 1 g/l of sediment or 3 mg/l of activated sludge from wastewater treatment increased the biodegradation potential which is believed to be the result of increased bacterial concentration in the test solution. Biodegradation was significantly slower in tests conducted in absence of oxygen. Assessments of the toxic properties of antibiotics by studying the influence on the biodegradation rates of 14C-aniline at different concentrations of antibiotics showed that no tests were conducted at toxic concentrations.

Biodegradability of metronidazole, olaquindox, and tylosin and formation of tylosin degradation products in aerobic soil--manure slurries.

The use of veterinary drugs (primarily antibiotics) in animal husbandry harbors the risk that these compounds end up in the farmland when manure is used as fertilizer. The biodegradability of three compounds, olaquindox (OLA), metronidazole (MET), and tylosin (TYL), was simulated in soil--manure slurries with 50 g of soil per liter. Supplemental batch sorption tests revealed that insignificant amounts of OLA and MET were located in the soil phase, whereas only 0.1 to 10% of the added amounts of TYL remained in the liquid phase. This may reduce the bioavailability and thus biodegradation rates of TYL. Unidentified metabolites of OLA and TYL and four known TYL metabolites were detected using HPLC. However, none of these substances were seen to persist in the biodegradation experiments, indicating that OLA and TYL most likely were mineralized in the experiments. Neither the use of sandy or clayey soil nor the use of 0, 1, or 10% (V/V) of manure added to these soils had a significant effect on the degradation rates. Degradation half-lives for the primary degradation were 3.3--8.1 days for TYL, 5.8--8.8 days for OLA, and 13.1--26.9 days for MET. Based on comparisons of results obtained with the benchmark chemical aniline and degradation half-lives of this compound in nature, it was assessed that results obtained with the current test method slightly overestimate real-world biodegradation rates.

Development of copepod nauplii to copepodites--a parameter for chronic toxicity including endocrine disruption.

Test compounds including natural hormones, endocrine disrupters, environmentally occurring compounds, and reference compounds were tested for acute toxicity and inhibitory effect on larval development in the copepod Acartia tonsa. Three compounds, 17alpha-ethinylestradiol, p-octylphenol, and tamoxifen, known for their differing effects on the vertebrate estrogen system, were potent inhibitors of naupliar development. Other estrogens, 17beta-estradiol, estrone, and bisphenol A, had little potency. Testosterone and progesterone did not inhibit development, but the antiandrogen flutamide had inhibitory effect. Juvenile hormone III was a potent inhibitor, as was expected based on the literature, whereas 20-hydroxyecdysone had no effect. 3,4-Dichloroaniline was inhibitory on development, whereas other control compounds, potassium dichromate and 3,5-dichlorophenol, did not inhibit development. Six of the 17 test compounds had 50% lethal concentration to 50% effective concentration (EC50) ratios higher than 10. The results suggest that naupliar development, as a parameter, is able to detect hormonal disrupters in addition to other chemicals that have other specific modes of action.

Environmental risk assessment of antibiotics: comparison of mecillinam, trimethoprim and ciprofloxacin.

The effects of mecillinam, trimethoprim and ciprofloxacin, antibiotics used in the treatment of urinary tract infections, on the aquatic environment were assessed. Mecillinam and ciprofloxacin were both readily biodegradable (primary degradation) in activated sludge, whereas trimethoprim persisted. The toxicity of these antibiotics towards sludge bacteria, a green alga, a cyanobacterium, a crustacean and a fish were investigated; both mecillinam and ciprofloxacin were highly toxic to the cyanobacterium Microcystis aeruginosa (EC50 in the range 5-60 microg/L). Risk characterization for the aquatic environment was performed for the three compounds by calculating the predicted environmental concentration (PEC) and the predicted no-effects concentration (PNEC). A PEC/PNEC ratio of <1 indicates that, with the present pattern of use, no environmental risk is expected. PEC/PNEC ratios of <1 for present usage in Europe were found for mecillinam and trimethoprim whereas a PEC/PNEC ratio >1 was found for ciprofloxacin.

Environmental risk assessment of antibiotics: comparison of mecillinam, trimethoprim and ciprofloxacin.

The effects of mecillinam, trimethoprim and ciprofloxacin, antibiotics used in the treatment of urinary tract infections, on the aquatic environment were assessed. Mecillinam and ciprofloxacin were both readily biodegradable (primary degradation) in activated sludge, whereas trimethoprim persisted. The toxicity of these antibiotics towards sludge bacteria, a green alga, a cyanobacterium, a crustacean and a fish were investigated; both mecillinam and ciprofloxacin were highly toxic to the cyanobacterium Microcystis aeruginosa (EC(50) in the range 5-60 Ìg/L). Risk characterization for the aquatic environment was performed for the three compounds by calculating the predicted environmental concentration (PEC) and the predicted no-effects concentration (PNEC). A PEC/PNEC ratio of <1 indicates that, with the present pattern of use, no environmental risk is expected. PEC/PNEC ratios of <1 for present usage in Europe were found for mecillinam and trimethoprim whereas a PEC/PNEC ratio >1 was found for ciprofloxacin.

The effect of dimethoate and cypermethrin on soil-dwelling beetles under semi-field conditions.

The effect of cypermethrin and dimethoate exposure on soil-dwelling beetles, in spring barley at different growth stages, of doses of up to eight times maximum field application rate has been investigated. Doses up to eight times maximum field application rate of cypermethrin did not have any acute effects on larger beetles, such as P. melanarius and C. erratus. Small beetles (A. bilineata, A. dorsale, B. lanpros, B. obtusum) were not harmed by doses up to two times maximum field application rate. T. hypnorum was affected at maximum field rate. Dimethoate at maximum field application rate harmed all species, but in particular the smaller species. When dimethoate was applied in high foliage density fields in the summer, very severe acute effects on spring breeding beetles were found. In the autumn, when only a low crop cover existed, this very high effect was not observed. The severe effect in the summer may be explained by the mode of action of dimethoate on 'old beetles'. The observed high toxic effect of dimethoate on spring breeders in the summer is expected only to have limited effect on the population, because the spring breeders at this time of the year have finished their egg depositing in the soil.

1-octanol/water distribution coefficient of oxolinic acid: influence of pH and its relation to the interaction with dissolved organic carbon.

The distribution of oxolinic acid (OA) between 1-octanol and buffers at a broad range of pH values was studied and found to decrease with increasing pH. The distribution coefficient to dissolved organic carbon (DOC), log DDOC, was estimated and compared with an experimentally derived log DDOC, showing the experimental value to be almost three orders of magnitude higher. Because only the neutral molecule is assumed to distribute to 1-octanol, the interaction with DOC is considered to be electrostatic in character.

Acute and chronic toxicity of veterinary antibiotics to Daphnia magna.

The acute and chronic toxicity of nine antibiotics used both therapeutically and as growth promoters in intensive farming was investigated on the freshwater crustacean Daphnia magna. The effect of the antibiotics metronidazole (M), olaquindox (OL), oxolinic acid (OA), oxytetracycline (OTC), streptomycin (ST), sulfadiazine (SU), tetracycline (TC), tiamulin (TI) and tylosin (TY) was tested in accordance to the ISO (1989) and OECD (1996) standard procedures. The acute toxicities (48-h EC50 value, mg/l) in decreasing order were OA (4.6), TI (40), SU (221), ST (487), TY (680) and OTC (approximately 1000). NOECs were 340 mg/l for TC and 1000 mg/l for M and OL. Toxic effect on reproduction occurred generally at concentrations, which were one order of magnitude below the acute toxic levels. The chronic toxicity (EC50 values, mg/l) in the D. magna reproduction test in decreasing order were TI (5.4), SU (13.7), TC (44.8) and OTC (46.2). The NOECs (mg/l) obtained in the reproduction test with OA, ST, TY and M were 0.38 for OA, 32 for ST, 45 for TY and 250 for M. The observed toxicity of OA to D. magna indicates that this substance, which is a commonly used feed additive in fish farms, has a potential to cause adverse effects on the aquatic environment.

Algal toxicity of antibacterial agents used in intensive farming.

The growth inhibiting effects of eight antibiotics used either therapeutically or as growth promoters in intensive farming on two species of micro algae, Microcystis aeruginosa (freshwater cyanobacteria) and Selenastrum capricornutum (green algae) were investigated. The effects of the antibiotics benzylpenicillin (penicillin G) (BP), chlortetracycline (CTC), olaquindox (O), spiramycin (SP), streptomycin (ST), tetracycline (TC), tiamulin (TI) and tylosin (TY) were tested in accordance with the ISO 8692 (1989) standard protocol. Algal growth was measured as increase in chlorophyll concentration by extraction with ethanol followed by measurement of fluorescence. Results were quantified in terms of growth rates using the Weibull equation to describe the concentration response relationship. The toxicity (EC50 value, mg/l) in alphabetic order were BP (0.006); CTC (0.05); O (5.1); SP (0.005); ST (0.007); TC (0.09); TI (0.003) and TY (0.034) for M. aeruginosa. BP (NOEC = 100); CTC (3.1); O (40); SP (2.3); ST (0.133); TC (2.2); TI (0.165) and TY (1.38) for S. capricornutum. In this investigation M. aeruginosa is found to be about two orders of magnitude more sensitive than S. capricornutum. It was observed that most of the compounds were unstable during the test period due to hydrolysis and photolysis.