Strategies to evaluate biodegradability: application to chlorinated herbicides

The biodegradability of nitrochlorinated (diuron and atrazine) and chlorophenoxy herbicides (2,4-D and MCPA) has been studied through several bioassays using different testing times and biomass/substrate ratios. A fast biodegradability test using unacclimated activated sludge yielded no biodegradation of the herbicides in 24 h. The inherent biodegradability test gave degradation percentages of around 20–30 % for the nitrochlorinated herbicides and almost complete removal of the chlorophenoxy compounds. Long-term biodegradability assays were performed using sequencing batch reactor (SBR) and sequencing batch membrane bioreactor (SB-MBR). Fixed concentrations of each herbicide below the corresponding EC₅₀ value for activated sludge were used (30 mg L^?1 for diuron and atrazine and 50 mg L^?1 for 2,4-D and MCPA). No signs of herbicide degradation appeared before 35 days in the case of diuron and atrazine and 21 days for 2,4-D, whereas MCPA was partially degraded since the early stages. Around 25–36 % degradation of the nitrochlorinated herbicides and 53–77 % of the chlorophenoxy ones was achieved after 180 and 135 days, respectively, in SBR, whereas complete disappearance of 2,4-D was reached after 80 days in SB-MBR.     

A new bioseed for determination of wastewater biodegradability: analysis of the experimental procedure

A new bioassay proposed in the patent P201300029 was applied to a pre-treated wastewater containing a mixture of commercial pesticides to simulate a recalcitrant industrial wastewater in order to determine its biodegradability. The test uses a mixture of standardized inoculum of the lyophilized bacteria Pseudomonas putida with the proper proportion of salts and minerals. The results highlight that biodegradation efficiency can be calculated using a gross parameter (chemical oxygen demand (COD)) which facilitates the biodegradability determination for routine water biodegradability analysis. The same trend was observed throughout the assay with the dehydrated and fresh inoculums, and only a difference of 5 % in biodegradation efficiency (E _f) was observed. The obtained results showed that the P. putida biodegradability assay can be used as a commercial test with a lyophilized inoculum in order to monitor the ready biodegradability of an organic pollutant or a WWTP influent. Moreover, a combination of the BOD₅/COD ratio and the P. putida biodegradability test is an attractive alternative in order to evaluate the biodegradability enhancement in water pre-treated with advanced oxidation processes (AOPs).     

Growth Inhibition by Nitrocompounds of Selected Uric Acid-Utilizing Microorganisms Isolated from Poultry Manure

The overall objective of this study was to evaluate the potential ability of nitrocompounds to reduce ammonia volatilization by inhibiting uric acid–utilizing microorganisms. Experiment I was conducted to evaluate the effects of nitrocompounds on the growth of uric acid–utilizing microorganisms isolated from poultry manure during six-hour incubation. There were five treatments: (1) control, (2) 50 mM nitroethane, (3) 50 mM nitroethanol, (4) 50 mM nitropropanol, and (5) 50 mM nitropropionic acid. Optical density values of nitrocompounds were significantly lower than that of control at two, four, and six hours. Plate counts of uric acid–utilizing microorganisms after six-hour incubation exhibited that nitrocompounds greatly reduced the growth of these microorganisms except for the nitroethane (P < 0.05). The nitropropanol and nitropropionic acid treatments showed significantly higher inhibitory effects compared to the nitroethanol. Experiments II and III were conducted to evaluate inhibitory effects of nitrocompounds on growth of uric acid–utilizing microorganisms compared to non-nitrocompounds such as ethanol, propanol, and propionic acid. Experiments II and III consisted of seven treatments: (1) control, (2) nitroethanol, (3) nitropropanol, (4) nitropropionic acid, (5) ethanol, (6) propanol, and (7) propionic acid. The incubation times of Experiments II and III were 6 and 24 h, respectively. The nitrocompounds were significantly more successful in inhibiting growth of uric acid–utilizing microorganisms compared to those non-nitrocompounds. These results suggest that nitrocompounds exhibit potential to reduce ammonia volatilization in poultry manure by inhibiting growth of uric acid–utilizing microorganisms.     

Relationship Between Biogenic Amines and Free Amino Acid Contents of Wines and Musts from Alentejo (Portugal)

The concentration of biogenic amines and free amino acids was studied in 102 Portuguese wines and 18 musts from Alentejo demarcated (D.O.C.) regions. Most wines were commercial, except for 38 monovarietals obtained by micro vinification. Musts from the varieties used to produce the latter wines were also studied. Both biogenic amines and free amino acids were analyzed by HPLC using fluorescence detection for their o-phthalaldehyde/fluorenylmethyl chloroformate (OPA/FMOC) derivatives. The most significant amines (average 10.8 mg/L for histamine+tyramine in red, and 7.4 mg/L for white wines) were found to be present at low levels and, although no important relationship between each individual biogenic amine could be obtained, the total amine content depends significantly on the assimilable amino acid content in wine.     

A two-generational reproductive toxicity study of zinc in rats

A two-generation reproductive toxicity study of zinc chloride (ZnCl₂) was conducted in rats. F_o male and female rats were administered 0.00 (control), 7.50 (low), 15.00 (mid) and 30.00 (high) mg/kg/day of ZnCl₂. Selected F₁ male and female rats were exposed to the same doses received by their parents (F_o). Exposure of F₀ parental rats to ZnCl₂ showed significant reduction in fertility, viability (days 0 and 4), and the body weight of F₁ pups from the high-dose group but caused no effects on litter size, weaning index, and sex ratio. Similarly, the continued exposure of F₁ parental rats to ZnCl₂ also reduced fertility, liter size, viability (day 0), and the body weight of F₂ pups within the high-dose group but caused no effects on weaning index and sex ratio. Exposure of ZnCl₂ to F₀ and F₁ parental males resulted in a significant reduction in their body weights, and the F₀ and F₁ parental females did not show any significant difference in their body weights compared to their control groups. However, the postpartum dam weights of both F₀ and F₁ female rats were significantly reduced compared to their controls. Exposure of ZnCl₂ to F_o and F₁ generation parental rats did not produce any significant change of their clinical signs as well as their clinical pathology parameters, except the alkaline phosphotase (ALK) level, which showed an upward trend in both sexes of both generations. Exposure of ZnCl₂ to F₀ rats resulted in a reduction of brain, liver, kidney, spleen and seminal vesicles weights of males and in the spleen and uterus of females. Similarly, exposure of F₁ rats to ZnCl₂ also resulted in reduction of brain, liver, kidney, adrenal, spleen, prostate and seminal vesicles weights of males and in spleen and uterus of females. ZnCl₂ exposure resulted in grossly observed gastro-intestianla (GI) tract, lymphoreticular/hematopoietic, and reproductive tract lesions in parental rats in both generations. Reduced body fat was also recorded in F₁ parental rats.     

Effects of glyphosate and foliar amendments on activity of microorganisms in the soybean rhizosphere

A field study was conducted to determine the effects of glyphosate on microbial activity in the rhizosphere of glyphosate-resistant (GR) soybean and to evaluate interactions with foliar amendments. Glyphosate at 0.84 kg ae ha^{? 1} was applied GR soybean at the V4–V5 development stages. Check treatments included a conventional herbicide tank mix (2003 study only) and no herbicides (hand-weeded). Ten days after herbicide application, a commercially available biostimulant and a urea solution (21.0% N) were applied to soybean foliage at 33.5 mL ha^{? 1} and 9.2 kg ha^{? 1}, respectively. Soil and plant samples were taken 0, 5, 10, 15, 20 and 25 days after herbicide application then assayed for enzyme and respiration activities. Soil respiration and enzyme activity increased with glyphosate and foliar amendment applications during the 2002 growing season; however, similar increases were not observed in 2003. Contrasting cumulative rainfall between 2002 and 2003 likely accounted for differences in soil microbial activities. Increases in soil microbial activity in 2002 suggest that adequate soil water and glyphosate application acted together to increase microbial activity. Our study suggests that general soil microbial properties including those involving C and N transformations are not sensitive enough to detect effects of glyphosate on rhizosphere microbial activity. Measurements of soil-plant-microbe relationships including specific microbial groups (i.e., root-associated Fusarium spp.) are likely better indicators of impacts of glyphosate on soil microbial ecology.     

Use of cholinesterase activity in monitoring chlorpyrifos exposure of steer cattle after topical administration

The aim of this work was to study the pharmacokinetic behavior and the inhibitory effect of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities of chlorpyrifos (CPF) in steer cattle after pour-on administration. Determination of cholinesterase activity in plasma and erythrocyte was carried out according to Ellman kinetic method. CPF was analyzed by gas chromatography. AChE was the predominant form of cholinesterase analyzed, with low levels of BChE in plasma. Following the treatment with CPF, the maximum inhibitory effect on AChE or BChE were 50.88 ± 11.57 and 42.66 ± 12.01%, respectively. The chlorpyrifos plasma concentrations observed were low and they presented a high variability. Chlorpyrifos peak plasma concentration (10.42 ± 4.76 μ g/L) was reached at 8.42 ± 13.97 h. The pesticide was not detected in plasma after 48 h post treatment. The values of area under the curve (AUC) were 118.48 ± 87.46 μ g· h/L and mean resistance time (MRT) were 13.38 ± 10.41 h. The pour-on exposure to the organophosphate chlorpyrifos significantly reduced AChE and BChE activity in steer cattle and the recovery was not reached on 50 days post-treatment.     

In vitro approaches to assess bioavailability and human gastrointestinal mobilization of food-borne polychlorinated biphenyls (PCBs)

This study reports on the potential for gastrointestinal (GI) mobilization and bioavailability of food-borne PCBs in humans. The development and validation of a GI simulator and operational protocols, developed in compliance with the requirements of German DIN 19738 risk assessment test procedure, are presented. Food, naturally contaminated with PCBs, was homogenized with simulated saliva fluid and shaken in the GI simulator with simulated gastric fluids (containing pepsin, mucine) for 2 h at 37°C. Afterwards, the simulated intestinal fluids (containing pepsin, mucine, trypsin, pancreatin, bile) were added and the mixture shaken for a further 6 h prior to centrifugation and filtration using Buchner funnels to separate the undigested GI residues from GI fluids. PCBs were recovered from GI residues and fluids by Soxhlet and liquid-liquid extraction respectively, cleaned up using silica-SFE, and analyzed by gas chromatography mass spectrometry detection (GC-MSD). Detailed studies with fish indicate variations in mobilization and bioavailability of Σ PCBs (28, 52, 101, 118, 153, 138 and 180). For example, the bioavailable fractions (fractions mobilized) in mackerel, salmon, crab and prawn were 0.77, 0.60, 0.54, and 0.72 respectively of the Σ PCBs initially present in these food samples. The bioavailable fraction was dependent on the physicochemical characteristics of the PCBs. In mackerel bioavailable fractions for individual PCB congeners ranged from 0.47–0.82, from 0.30–0.70 in salmon, 0.44–0.64 in crab and in prawn from 0.47–0.77. Future studies will focus on understanding better, the variability in bioavailable fractions to be expected for different foodstuffs, in addition to tissue culture techniques using human gut cell lines to investigate a simultaneous mobilization and absorption of food-borne PCBs.     

Fate in soil of 14C-sulfadiazine residues contained in the manure of young pigs treated with a veterinary antibiotic

The fate of ¹⁴C-labeled sulfadiazine (¹⁴C-SDZ) residues was studied in time-course experiments for 218 days of incubation using two soils (A_p horizon of loamy sand, orthic luvisol; A_p horizon of silt loam, cambisol) amended with fresh and aged (6 months) ¹⁴C-manure [40 g kg^?1 of soil; 6.36 mg of sulfadiazine (SDZ) equivalents per kg of soil], which was derived from two shoats treated with ¹⁴C-SDZ. Mineralization of ¹⁴C-SDZ residues was below 2% after 218 days depending little on soil type. Portions of extractable ¹⁴C (ethanol-water, 9:1, v/v) decreased with time to 4–13% after 218 days of incubation with fresh and aged ¹⁴C-manure and both soils. Non-extractable residues were the main route of the fate of the ¹⁴C-SDZ residues (above 90% of total recovered ¹⁴C after 218 days). These residues were high immediately after amendment depending on soil type and aging of the ¹⁴C-manure, and were stable and not remobilized throughout 218 days of incubation. Bioavailable portions (extraction using CaCl₂ solution) also decreased with increasing incubation period (5–7% after 218 days). Due to thin-layer chromatography (TLC), 500 μg of ¹⁴C-SDZ per kg soil were found in the ethanol-water extracts immediately after amendment with fresh ¹⁴C-manure, and about 50 μg kg^?1 after 218 days. Bioavailable ¹⁴C-SDZ portions present in the CaCl₂ extracts were about 350 μg kg^?1 with amendment. Higher concentrations were initially detected with aged ¹⁴C-manure (ethanol-water extracts: 1,920 μg kg^?1; CaCl₂ extracts: 1,020 μg kg^?1), probably due to release of ¹⁴C-SDZ from bound forms during storage. Consistent results were obtained by extraction of the ¹⁴C-manure-soil samples with ethyl acetate; portions of N-acetylated SDZ were additionally determined. All soluble ¹⁴C-SDZ residues contained in ¹⁴C-manure contributed to the formation of non-extractable residues; a tendency for persistence or accumulation was not observed. SDZ's non-extractable soil residues were associated with the soluble HCl, fulvic acids and humic acids fractions, and the insoluble humin fraction. The majority of the non-extractable residues appeared to be due to stable covalent binding to soil organic matter.     

The fate of recombinant plasmids during composting of organic wastes

Composting was investigated as a means for safe disposal of organic waste containing bacteria that carry transgenes in recombinant plasmids. To generate model recombinant plasmids, a mobile IncQ plasmid, RSF1010, and a non-mobile plasmid, pGFP, were genetically modified to carry a DNA segment encoding both green fluorescent protein and kanamycin resistance and were designated as RSF1010-GFPK and pGFPK. Escherichia coli (E. coli) C600 harboring these plasmids were inoculated into chicken manure specimens that were placed in compost at 20 and 60 cm from the bottom of a 1.0-m high compost bin. Control specimens were held at ambient temperature. By day 10, compost temperatures at the lower and upper levels of the bin had reached 45.3 and 61.5°C, respectively, and at both levels the target E. coli had been inactivated and the plasmids had lost their capacity to be transformed or mobilized. Furthermore, based on real time Polymerase chain reaction (PCR), the transgene fragments along with the host chromosomal DNA fragment from specimens at the upper level had been degraded beyond the detection limit. However, at the lower level where temperatures remained below 48°C these fragment persisted to day 21. At ambient temperatures (0–8°C), the E. coli, plasmids and the transgene fragments persisted in manure specimens throughout the 21 day test period. The study showed the potential for composting as a safe procedure for disposal of bacteria carrying transgenes in recombinant plasmids.