Bioavailability to rats of bound [14C] pirimiphos-methyl in stored wheat.

Stored wheat treated with radiolabelled pirimiphos-methyl (0-2-diethyl-amino-6-methyl-pyrimidin-4-yl 0,0-dimethyl phosphorothioate) formed bound (nonextractable) 14C residues. Supercritical fluid extraction, gas chromatography and mass spectrometric techniques were used to identify and quantitate the 14C bound residues in wheat grains. The amount of bound 14C residues present after 28 weeks of storage was about 9.9% of the applied radioactivity. Pirimiphos-methyl accounted for 80% of the bound residue. Grain-bound residues were fed to rats for 5 days. After a total period of 8 days a substantially large percentage of the administered bound 14C residues (72.9%) was eliminated in urine while feces contained only 17.9%. Bound pirimiphos-methyl in wheat grain was metabolized in rats by processes involving hydrolysis, N-dealkylation and 0-demethylation. The results indicate that wheat-bound residues of pirimiphos-methyl are highly bioavailable to the rat and may possess a toxicological potential as manifested by a significant reduction in body weight gain.     

Bioavailability to rats and toxicity in mice of carbofuran residues bound to faba beans.

14C-carbofuran penetrated readily into seeds of Vicia faba and the rate of penetration was found to be dose dependent. The percentage of bound residues was generally low and did not exceed 3% of the applied dose. When the bound residues were fed to rats 46% of the radioactivity was eliminated via CO2 and urine, while tissues contained 25%. Carbofuran phenol and 3-hydroxy carbofuran represented the main metabolites in the urine. These data indicate that bean-bound carbofuran residues are highly bioavailable to rats. Feeding mice with bound carbofuran residues for 90 days led to inhibition of erythrocyte cholinesterase activity after 30 days (35-40%) while the plasma enzyme remained unaffected. Serum transaminases and blood urea nitrogen were significantly elevated, indicating injury to hepatic and renal structures. The results strongly suggest that the bound residues can induce adverse biological effects in mice.     

Bioavailability of bound 14C residues in rats from bean plants treated with 14C-deltamethrin

Bean plants were treated with deltamethrin labeled with ¹⁴C at the methyl or benzylic position. The aerial portion of the plants was exhaustively extracted with solvents and the extracted material containing bound ¹⁴C residues was fed to rats. After 4 days 60% and 53% of the dose was excreted in feces and 31% and 20% in the urine from rats fed extracted bean plants treated with deltamethrin labeled at the methyl and benzylic position, respectively. The data demonstrated that bound residues in bean plants treated with deltamethrin may be bioavailable in rats.     

Bioavailability, biological activity and characterization of bound residues of diflubenzuron in wheat.

Wheat grain was treated with radiolabeled diflubenzuron at 100 ppm and stored for various periods; up to 6 months. The grain was surface washed, Soxhlet-extracted with methanol, and the residues determined. A relative constant amount of bound residues (4%), i.e., non-extractable radioactivity, was found 4 months after application and remained constant. More than 97% of the extractable radioactivity in the grain after 6 months was identified as diflubenzuron. When the bound residues were fed to rats, 47% of the administered dose was eliminated via the urine and the remainder via feces within 96 h. Diflubenzuron was the major component in the urine. Adding bound residues to housefly media resulted in a dose-dependent mortality of housefly pupae. Bound residues were biologically active, preventing the emergence of adult houseflies. Supercritical fluid extraction of the bound residues extracted 92% and 96% of the radioactivity associated with grain and feces, respectively. Only diflubenzuron was present in these extracts. The bioavailability and biological activity of bound residues of diflubenzuron have been demonstrated and the identity of the radioactivity was shown to be parent compound. Based on these findings, bound pesticide residues can no longer be ignored or overlooked in the evaluation of pesticide residues and their possible toxicological implications.     

Bioavailability to rats and toxicological potential in mice of bound residues of malathion in beans.

Under conditions of local practice, the application of 2,3-succinate-14C-malathion on beans led to the formation of 17-18% of bound 14C-residues after 30 weeks. When fed to rats, 75% of these residues became bioavailable after 2 days with the major part, excreted via expired air (8%) and urine (60%). The main radioactive metabolites detected in urine were malathion monocarboxylic acid and malathion dicarboxylic acid. Feeding of bound residues to mice (1.8 ppm in feed) for 90 days resulted in a reduction in body weight gain after 60 days and inhibition of erythrocyte cholinesterase activity after 90 days. Increased levels of serum glutamic oxaloacetic transaminase and alkaline phosphatase were also observed. The results strongly suggest that bean-bound malathion residues can cause adverse biological effects in mice.     

Biological activity and bioavailability of grain bound 14C-malathion residues in rats.

Paddy (unmilled rice), milled rice and maize-bound 14C residues were prepared using 14C-succinate-labelled malathion at 10 and 152 ppm. After 3 months, the bound residues accounted for 12%, 6.5% and 17.7% of the applied dose in paddy, milled rice and maize respectively in the grains treated at 10 ppm. The corresponding values for the 152 ppm were 16.6%, 8.5% and 18.8%. Rats fed milled rice - bound 14C-residues eliminated 61% of the 14C in the faeces and 28% in the urine. The corresponding percentages for paddy and maize were 72%, 9% and 53%, 41% respectively; indicating that bound residues from milled rice and maize were moderately bioavailable. When rice-bound malathion residues (0.65 ppm in feed) were administered to rats in a 5 week feeding study, no signs of toxicity were observed. Plasma and RBC cholinesterase activities were slightly inhibited: blood urea nitrogen was significantly elevated in the test animals. Other parameters examined showed no or marginal changes.     

Bioavailability and toxicological potential of sunflower-bound residues of 14C-chlorpyrifos insecticide in rats

Sunflower plants were treated with ¹⁴C-chlorpyrifos under conditions simulating local agricultural practice. Residues present in the oil, methanol extract and cake of the treated sunflower seeds were 7.2, 2.8, and 12 ppm, respectively. When rats fed on sunflower cake containing bound residues for three days, the animals eliminated 46 % of the radioactivity in urine, 25 % in feces and 10 % in the expired air. A further bioavailable amount of 8 % was found in selected organs indicating that the bound residues were highly bioavailable. Chromatographic analysis of urine extract revealed the presence of the parent compound, its oxon, desethyl chlorpyrifos and desethyl chlorpyrifos oxon as free metabolites in addition to a conjugated metabolite. It was liberated by acid hydrolysis and identified as 3,5,6-trichloro-2-hydroxypyridine. Bound residues were found to have biological effects such as inhibition of rat plasma ChE, elevations of liver parameters (ALT, AST, and ALP), decrease in total protein and albumin content suggesting a hepatotoxic potential. A significant increase in the values of creatinine, urea, cholesterol, triglycerides and significant decrease in Catalase and Glutathion-S-Transfrase were observed in treated rats.     

Bioavailability and biological activity of bound residues of radiolabelled pirimiphos-methyl in maize grains.

Using a 14C-labelled pirimiphos-methyl preparation, the percentage of pirimiphos-methyl residues bound to maize grains after 32 weeks of storage was 13% of the applied dosage, or 38% of total terminal residues. Evidence is presented to show that bound residues of pirimiphos-methyl are bioavailable to the rat: 30%, 2% and approx. 6% of radioactivity were measured in urine expired air, and some organs respectively. A major portion of radioactivity (55%) was eliminated through faeces. Grain-bound pirimiphos-methyl residues (generated after storing whole maize grains with pirimiphos-methyl at concentrations of 10 ppm and 100 ppm) were administered to albino rats for 12 weeks. Body and organ weights, enzyme activities and blood chemistry were tested. There was a significant reduction in body weight gain in female rats. Also a significant reduction in blood cholinesterase activity was observed in both male and female rats fed on grain bound pirimiphos-methyl residues at two dose levels. The white blood cell count increased significantly in male rats fed on the high dose. No significant changes were observed in the other blood chemistry parameters tested. The results indicate that maize-bound pirimiphos-methyl residues can exert adverse biological effects in the rat.     

Bioavailability and toxicological potential of sunflower-bound residues of (14)C-chlorpyrifos insecticide in rats

Sunflower plants were treated with (14)C-chlorpyrifos under conditions simulating local agricultural practice. Residues present in the oil, methanol extract and cake of the treated sunflower seeds were 7.2, 2.8, and 12 ppm, respectively. When rats fed on sunflower cake containing bound residues for three days, the animals eliminated 46 % of the radioactivity in urine, 25 % in feces and 10 % in the expired air. A further bioavailable amount of 8 % was found in selected organs indicating that the bound residues were highly bioavailable. Chromatographic analysis of urine extract revealed the presence of the parent compound, its oxon, desethyl chlorpyrifos and desethyl chlorpyrifos oxon as free metabolites in addition to a conjugated metabolite. It was liberated by acid hydrolysis and identified as 3,5,6-trichloro-2-hydroxypyridine. Bound residues were found to have biological effects such as inhibition of rat plasma ChE, elevations of liver parameters (ALT, AST, and ALP), decrease in total protein and albumin content suggesting a hepatotoxic potential. A significant increase in the values of creatinine, urea, cholesterol, triglycerides and significant decrease in Catalase and Glutathion-S-Transfrase were observed in treated rats.     

Bioavailability and biological activity of wheat-bound chlorpyrifos-methyl residues in rats.

Wheat grain was treated with 14C-chlorpyrifos-methyl to generate bound residues for determining their bioavailability to rats. In a parallel experiment, bound residues were prepared with non-labelled chlorpyrifos-methyl to determine possible adverse effects in rats fed the grain-bound residue for 28 and 90 days. Two dose levels of 10 and 50 ppm were initially used on the grain. The 10 ppm led to the formation of 25.1% bound residues (2.51 ppm) after 6 months as determined by radiomeasurement. The higher dose was assumed to form 12.55 ppm bound residues. When 14C-bound residues were fed to male rats for 24 hours, the animals eliminated 75% of the radioactivity in urine, 7% in expired air and 8% in faeces after 3 days, indicating that the bound residues were highly bioavailable. A further "bioavailable" amount (4%) was found in selected organs.     

Bioavailability and toxicological potential of lentil-bound residues of malathion in rats.

Lentil grains treated with malathion and stored under laboratory conditions for 12 months formed bound residues. Bioavailability and the effects of lentil-bound residues of malathion in rats were studied. The amount of bound residues in lentils treated with 14C-malathion at 10 ppm and 50 ppm gradually increased to 9.52% and 13.01% of the initially applied doses after 12 months. When rats were fed these 14C-bound residues, radioactivity excreted in urine accounted for 34.49% of the administered dose. In feces, 26.15% of given dose was methanol-extractable while 18.67% was determined as nonextractable. Various tissues including liver, kidney, fat and lungs contained 8.93% while radioactivity in expired air (14CO2) was low (1.51%). The results indicate that lentil-bound malathion residues are highly bioavailable to rats. Analysis of the lentil material containing bound residues indicated that the main compound was malathion. Lentil-bound malathion residues were administered to albino rats at 0.95 and 6.51 ppm in the feed for 3 months. Body weights were determined during and at the end of the experiment. Terminal organ weights were also determined and a number of blood chemistry parameters were examined. A significant reduction in serum cholinesterase activity and an increase in blood urea nitrogen and in white cell count suggest a toxocological potential of the bound residues.     

Studies on bound 14C-prometryn residues in soil and plants

A high-temperature distillation technique was developed for determining and chemically identifying the bound (nonextractable) residues of ¹⁴C-prometryn in an organic soil and plants. A considerable portion of the bound ¹⁴C residues in the incubated organic soil was identified as prometryn. These residues were absorbed by plants grown in the soil. Hono-and di-$\text{N}$-dealkylated metabolites of prometryn were present in the plant bound ¹⁴C residues and a major portion of bound residues as associated with lignin. Soil-bound ¹⁴C residues were also released from soil by microbes. The bound ¹⁴C residues in soil were associated with humin, humic acid, and fulvic acid fractions. Thermoanalytical methods were used to obtained information on the nature and location of ¹⁴C bound residues in soil and humic materials.     

Accumulation of 14C-trinitrotoluene and related nonextractable (bound) residues in Eisenia fetida

To determine if trinitrotoluene (TNT) forms nonextractable residues in earthworms and to measure the relative degree of accumulation as compared to TNT and its deaminated metabolites, Eisenia fetida was exposed to ¹⁴C-TNT using dermal contact to filter paper or exposure to soil. Nonextractable residues made up 32-68% of total body burden depending on exposure media and depuration time. Parent TNT accounted for less than 3% of radioactivity, while ADNTs accounted for 7-38%. Elimination half-lives were 61-120 h for TNT, ADNTs, and DANTs, which was significantly lower than the half-lives found for nonextractable residues, 201-240 h. However, over 80% of the nonextractable residue was solubilized using weak acid (pH 2). Based on our findings that TNT accumulation occurs primarily as nonextractable residues, which have a longer half-life, and that nonextractable residues can be solubilized, we propose that nonextractable residues could be used as a selective biomarker for assessing TNT contamination.     

A model for the formation and degradation of bound residues of the herbicide 14C-isoproturon in soil.

The humic monomer catechol was reacted with 14C-isoproturon and some of its metabolites, including 14C-4-isopropylaniline, in aqueous solution under a stream of oxygen. Only in the case of 14C-4-isopropylaniline, incorporation in oligomers, in fulvic acid-like polymers, and in humic acid-like polymers was observed. The main oligomer was identified by mass spectrometry as 4,5-bis-(4-isopropylphenylamino)-3,5-cyclohexadiene-1,2-dione. Oligomers and polymers containing bound 14C-4-isopropylaniline were subjected to biodegradation studies in a loamy agricultural soil during 55 days by quantifying 14CO2 evolved. In all cases, significant mineralization rates could be determined, which, however, were much smaller than those of free 14C-4-isoproturon and free 14C-4-isopropylaniline in the same soil.     

Aged and bound herbicide residues in soil and their bioavailability. Part 2: Uptake of aged and non-extractable (bound) [carbonyl-14C]methabenzthiazuron residues by maize

[Carbonyl-14C]methabenzthiazuron (MBT) was applied to growing winter wheat in an outdoor lysimeter. The amount applied corresponded to 4 kg Tribunil/ha. 140 days after application the 0-2.5 cm soil layer was removed from the lysimeter. This soil contained about 40% of the applied radioactivity. Using 0,01 M CaCl2 solution or organic solvents, the extractable residues were removed from the soil. The bioavailability of the non-extractable as well as aged residues remaining in the soil was investigated in standardized microecosystems containing 1.5 kg of dry soil. During a 4 weeks period the total uptake (4 maize plants/pot) amounted up to 3.6; 2.2; and 0.9% of the radioactivity from soils containing aged MBT residues, MBT residues non-extractable with 0.01 M CaCl2 or MBT residues non-extractable with organic solvents, respectively. About 20% of the radioactivity found in maize leaves represented chromatographically characterized parent compound. At the end of the plant experiment the soil was extracted again with 0.01 M CaCl2 and with organic solvents. The soil extracts and also the organic phases obtained from the aqueous fulvic acid solution contained unchanged parent compound.     

Terbufos residues in wheat and barley

A granular formulation of terbufos (Counter 15G) was added in-furrow at time of planting of wheat and barley. Foliage collected at several times was analyzed for total terbufos residues as terbufoxon sulfone. Maximum residues from application of 1.5 and 3.0 kg/ha were 7.4 and 10.6 ppm, respectively, in wheat foliage samples collected 10 days postseeding. Wheat foliage collected at 53 days postseeding had residues averaging 0.32 and 0.58 ppm from the 1.5 and 3.0 kg/ha applications, respectively. In 1985 residues in barley were consistently less than in wheat in 1985 with 4.4 and 7.0 ppm detected in foliage collected 10 days post application from the 1.5 and 3.0 kg/ha applications, respectively and 0.21 and 0.34 ppm detected at 53 days. Grain samples had 0.01 ppm or less residue at harvest. Straw samples had up to 0.75 ppm total terbufos residues at harvest.     

Pesticide persistence and bound residues in soil--regulatory significance

The paper discusses key aspects of the European Union (EU) regulatory policy related to the persistence and bound residues of agricultural pesticide active substances in soil. This is examined in the context of the EU Authorisations Directive (91/414/EEC) which will gradually replace existing national systems of agricultural pesticide regulation within EU Member States. Discussion is concentrated on this directive, looking in particular at the Uniform Principles therein and the possible ways that these decision-making guidelines could be developed into a regulatory framework. The aim in this process of negotiated development is to identify any questions or data requirements that will be needed for persistent pesticides or soil bound residues, over and above those generally required for all compounds. The present EU regulatory position on soil non-extractable or bound residues is examined and possible future improvements to the system are described and discussed.     

Formation and release of residues of the 14C-labelled herbicide isoproturon and its metabolites bound in model polymers and in soil

The humic monomer catechol was reacted with (14)C-isoproturon and some of its metabolites, including (14)C-4-isopropylaniline, in aqueous solution under a stream of oxygen. Only in the case of (14)C-4-isopropylaniline, incorporation in oligomers, in fulvic acid-like polymers, and in humic acid-like polymers by covalent bonds was observed. The main oligomer was identified by mass spectrometry as a trimer, 4,5-bis-(4-isopropylphenylamino)-3,5-cyclohexadiene-1,2-dione. Biomineralization of (14)C-compounds to (14)CO(2) in a loamy soil and release of (14)C from soil columns into percolate water decreased in the order: free isoproturon >free 4-isopropylaniline>fulvic acid-like polymers>trimer>humic acid-like polymers. In soil columns, a small but measurable migration of (14)C from polymers from upper to deeper soil layers could be detected; most of this (14)C was bound again in a non-extractable form. It is concluded that aniline-derived pesticides bound in soil by covalent binding may not be fully undegradable, nor fully immobile.     

In vitro release of bound (nonextractable) 14C residues from plants by corn leaves bomogenates

Corn leaves homogenates were found to release bound (nonextractable) ¹⁴C residues from the aerial portion of matured corn plants. The 22,000 g pellet and 10,000 g supernatant fractions were the most active in releasing the bound ¹⁴C residues. The released ¹⁴C residues comprised mainly 2-OH analogues of the $\text{N}$-monodealkylated analogues of atrazine. It is suggested that the enzymatic system in plants may cause metabolic conversion of bound residues.     

Causes of phytotoxicity of metsulfuron-methyl bound residues in soil

The bioavailability and phytotoxicity of bound residues of metsulfuron-methyl were studied using 14C-labeling and bioassay with oil rape (Brassica napus L.). Soil bound residues at the concentration of 0.27 and 0.53 nmol g(-1) resulted in significant inhibition of oil rape seedling growth. The biologically active component of the bound residues was identified to be metsulfuron-methyl parent compound. Other metabolites, including the hydrolysis product 2-methylformate-benzenesulfonamide, showed no toxicity to the test species. This study suggests that residues of metsulfuron-methyl bound previously to the soil matrix could be again released upon planting. The phytotoxicity of metsulfuron-methyl bound residues was mainly caused by the metsulfuron-methyl parent compound that became available during plant growth.