RESIDUE AND ANALYTICAL ASPECTS
Carbosulfan, 2,3-dihydro-2,2-dimethylbenzofuran-7-yl (dibutylaminothio)methylcarbamate is a broad-spectrum carbamate pesticide used on a variety of crops, but mainly on citrus fruits, and this use is the focus of the present evaluation. Carbosulfan was first evaluated by the 1984 JMPR, which recommended a temporary ADI and a temporary MRL of 2 mg/kg for citrus fruits. The temporary ADI was converted to an ADI of 0-0.01 mg/kg bw by the 1986 JMPR.
Because information required by the 1984 and 1991 Meetings had not been provided, the 1993 JMPR recommended withdrawal of the proposed TMRLs for carbofuran and carbosulfan in citrus fruits. The 1993 Meeting was informed that additional studies were under way. Carbosulfan was subsequently scheduled for periodic review by the FAO Panel in 1997. New studies on citrus fruit have been reviewed by the Meeting, together with supporting data.
Carbofuran is a major metabolite of carbosulfan as well as being itself a pesticide. The present periodic review of carbosulfan includes estimates of maximum residue levels, an STMR and STMR-Ps for carbosulfan per se resulting from its use on citrus fruit. The concurrent review of carbofuran includes estimates to accommodate residues of carbofuran and 3-hydroxycarbofuran resulting from the use of carbosulfan on citrus fruit.
Metabolism studies on rats and goats were available. The distribution, excretion and fate of carbosulfan was investigated in rats by oral gavage administration of dibutylamine- or phenyl -labelled [14C] carbosulfan at low (4 mg/kg) or high (30 mg/kg) dosing levels. About 66-88% was eliminated in the urine, 5-22% in the faeces and 10 to 17% as CO2 from the dibutylamine (DBA) label but none from the phenyl label. Up to about 2% remained in the carcase. Eighty to 90% was excreted within 24 to 48 hours of dosing at the lower dose and within 72 hours or so at the higher dose. The main excreted compounds identified from the phenyl label in decreasing order were the 7-phenol, 3-keto-7-phenol, 3-hydroxycarbofuran, 3-hydroxy-7-phenol and carbosulfan, with minor residues of 5-hydroxycarbofuran, 3-ketocarbofuran, carbofuran, 3-ketocarbosulfan sulfone, 3-hydroxycarbosulfan and 3-ketocarbosulfan. From the DBA label DBA, hydroxy-DBA, CO2 and carbosulfan were found in decreasing order. No major sex differences were observed. Higher residues of 14C were found in tissues from the DBA label than from the phenyl. This was attributed to incorporation of the DBA moiety into natural stored fat by oxidation, N-dealkylation or deamination and further oxidation to fatty acids.
The metabolites are consistent with metabolic routes which include a series of hydrolyses, oxidations and conjugations. A main indicated route includes hydrolysis to the 7-phenol, oxidation to 3-hydroxy-7-phenol and further to 3-keto-7-phenol, and conjugation as sulfates or glucuronides. Another route involves oxidation to 3-hydroxycarbosulfan which may be hydro lysed to 3-hydroxycarbofuran or oxidized further to 3-ketocarbosulfan, which in turn may be oxidized again to 3-ketocarbosulfan sulfone or hydrolysed to 3-ketocarbofuran. The 3-hydroxycarbofuran or 3-ketocarbofuran may be hydrolysed to their phenols before conjugation. Hydrolysis also results in the release of DBA which may be oxidized at different carbons to hydroxydibutylamines. The authors also postulate the N-dealkylation/deamination and oxidation to fatty acids which may be incorporated in natural fats as described above, or result in the release of CO2 by the citric acid cycle as indicated by the detection of radiolabelled CO2.
Lactating goats were dosed with either phenyl- or DBA-labelled carbosulfan for 7 days at levels corresponding to approximately 25 ppm in the diet. Samples of urine, faeces, milk and tissues were analysed. As in rats, most of the 14C was eliminated in the urine, approximately 82% of the phenyl label and 68% of the DBA label. About another 7% and 3% respectively were eliminated in the faeces. Approximately 0.2% of the dose (0.04-0.09 mg/kg carbosulfan equivalent) was found in the milk, 0.02% (0.06 mg/kg) in liver and 0.01% (0.2 mg/kg) in kidney from the phenyl label, but less than 0.01% (£ 0.01 mg/kg) in muscle or fat. Because of these low levels, the identification of 14 C residues in muscle and fat from the phenyl label was not attempted.
The residues were higher from the DBA label: 2.3% (0.3-0.94 mg/kg) in the milk, 0.34% (1.13 mg/kg) in the liver, 0.04% (0.75 mg/kg) in the kidneys, 0.08% (0.18 mg/kg) in the muscle, and 0.15% (1.2 mg/kg) in the omental fat. The higher levels were attributed to incorporation into natural body constituents such as carbohydrates and proteins. The detection of radioactivity in fatty acids, amino acids, triglycerides and amines was consistent with that hypothesis.
A series of extractions, partitions, pH adjustments and acid or enzymatic hydrolyses were used to separate metabolites for comparison with authentic standards by HPLC, TLC, GC-MS, HPLC-MS and size-exclusion chromatography. From the phenyl label 98.6% of the TRR was extractable from milk, 37.3% from liver and 62% from kidney.
The major metabolites identified in milk, liver and kidney from the phenyl label were 3-hydroxycarbofuran and the 3-keto-7-phenol, 3-hydroxy-7-phenol and 7-phenol, accounting for approximately 94.4% of the TRR in milk (reaching a plateau after about 2 days), 32.7% in liver (37.1% for all identified residues) and 52% in kidney (59.6% for all identified residues). 3-hydroxycarbofuran (34.2% of the TRR) and the 3-keto-7-phenol (29.9% of the TRR) predominated in the milk, 3-hydroxycarbofuran in kidney (21.5% of the TRR) and the 3-hydroxy-7-phenol in liver (15.6% of the TRR). Minor identified residues included 5-hydroxycarbofuran, 3-ketocarbofuran, TV-hydroxymethyl-carbofuran, carbofuran, carbosulfan, 3-ketocarbosulfan sulfone, 3-hydroxycarbosulfan and carbosulfan sulfone. None of these exceeded 4% of the TRR in milk, liver or kidney. Carbosulfan and carbofuran were detected only at very low levels in the milk and tissues (0.001 mg/kg).
Although only 37.1% of the phenyl label radioactivity was extractable from the liver with the initial solvent extraction, enzymatic and HCl hydrolysis allowed further characterization. Unidentified radioactivity was characterized as very polar (10.4% of the TRR), protein-associated (22.6% of the TRR) or unextractable (12.7% of the TRR).
In the kidney very polar unidentified metabolites accounted for 18.4% of the TRR, with another 17.4% characterized, but not identified.
Residue levels were much higher from the DBA label and this was attributed largely to incorporation into natural products as in rats. Residues in the fat and muscle from the phenyl label were too low for identification or characterization, but were high enough with the DBA-labelled samples (1.3 and 0.2 mg/kg carbosulfan equivalent in fat and muscle respectively). Approximately 80.1% of the DBA TRR (0.6 mg/kg carbosulfan equivalent) in milk and 90% in fat was organo-extractable, but only 45% in liver, slightly more than with the phenyl label. Approximately 70% of the TRR was extractable from kidney and 52% from lumbar muscle.
Residues in milk and tissues from the DBA label consisted mainly of aminobutanols, dibutylamine-related compounds, material incorporated into natural constituents (fatty acids, amino acids, carbohydrates, triglycerides etc.), amines (conjugated, non-conjugated and bound) and polar water-soluble metabolites. In milk aminobutanols accounted for approximately 30% of the TRR and another 30% was found in natural constituents. 87% of the TRR in fat, 32% in muscle and 30% in liver was found in natural constituents. In liver another 21% was in the form of aminobutanols or dibutylamine and related compounds. In kidney 24% of the TRR was characterized as non-conjugated amines, approximately 19% as polar water-soluble metabolites, and 14% as natural constituents.
The main metabolic routes in goats are similar to those in rats, starting with hydrolysis either directly to the 7-phenol or to carbofuran and dibutylamine. The 7-phenol is oxidized progressively to the 3-hydroxy-7-phenol and 3-keto-7-phenol and carbofuran to 3-hydroxy-or 5-hydroxycarbofuran. The 3-hydroxycarbofuran may be oxidized to 3-ketocarbofuran and each of these hydrolysed to the corresponding phenol. Dibutylamine may be oxidized to 4-(butylamino)butanol and further to the corresponding butanoic acid, or undergo a series of reactions to form butylamines and butanols. The degradation of DBA may also lead to incorporation into fatty acids, amino acids and carbohydrates and presumably through the citric acid cycle to CO2, but CO2 was not trapped.
The minor residues derived from the phenyl-labelled compound also indicate a subsidiary metabolic route in which the carbamate structure is retained with either direct oxidation to the sulfone or via 3-hydroxycarbosulfan and 3-ketocarbosulfan to 3-ketocarbosulfan sulfone.
Plant metabolism. Metabolism studies with both phenyl- and DBA-labelled carbosulfan were conducted in the field on navel oranges with spray application at a nominal rate of 0.5 g ai/l. Orange samples were taken at 0, 7, 15 and 30 days and leaves at 0 and 30 days. Oranges were rinsed and samples of peel rinse, peel, pulp and juice were analysed by HPLC, TLC, MS and LSC. The TRR in whole oranges amounted to 0.81 and 0.7 mg/kg carbosulfan equivalent from the phenyl and DBA labels on day 0 to 0.78 and 0.59 mg/kg on day 30.
Nearly all of the residue in the whole fruit was in or on the peel (99.9% of the phenyl 14 C, 99.6 of the DBA) on day 0 and these proportions remained essentially unchanged even after 30 days. Almost all of the residue was on the peel surface on day 0 (95.8% of the phenyl TRR, 93.9% of the DBA), but by 30 days more of the residue had penetrated into the peel (45.9% of the 14 C from the phenyl label and 41.5% from the DBA). No more than 0.3% of the TRR (<0.01 mg/kg carbosulfan equivalent) from both labels was in the pulp or juice over the 30-day period. More than 90% of the TRR in rinsed peel from both labels was extractable throughout the 30 days with the proportion of polar and conjugated material increasing with time, especially that from the DBA label where it reached 57% by day 30.
The peel rinses and extracts were examined to identify the residues. Residues from the phenyl label after 30 days as a proportion of the TRR were carbosulfan 40.1%, carbofuran 33.9%, carbosulfan sulfone 3.1%, 3-hydroxycarbofuran and 3-keto carbofuran 2% each and N-hydroxymethyl-carbofuran, dicarbofuran sulfide and the 7-phenol less than 2% each, making a total of 83.7% of the TRR. From the DBA label carbosulfan and DBA accounted for 31.2 and 58.2% of the TRR respectively. This is consistent with the primary metabolic cleavage of the two N-S bonds to form carbofuran and DBA. Some oxidation to carbosulfan sulfone occurs before cleavage of these bonds and a minor route resulted in the formation of dicarbofuran sulfide. The rest of the metabolism is effectively that of carbofuran. This includes direct oxidation to the 7-phenol or retention of the intact carbamate with oxidation at the N-methyl to form N-hydroxymethylcarbofuran or on the ring to form 3-hydroxycarbofuran which may be further oxidized to 3-ketocarbofuran.
The only metabolite found in oranges which was not also identified in goats was dicarbofuran sulfide at very low levels. The other notable difference between plants and animals is that only very low levels of intact carbamates were detected in animals (apart from 3-hydroxycarbofuran at exaggerated feeding levels), whereas they were the main residues in plants after 30 days.
Environmental fate. Although limited information was available on photolysis in soil and water, other environmental studies noted as being necessary in the 1995 JMPR report (Section 2.5.2) were not provided. The degradation of carbosulfan in dry soil and soil at 70% water capacity exposed to a sun lamp was investigated with phenyl- and DBA-labelled [14C] carbosulfan. The spectral characteristics of the sun lamp were not reported, nor was the temperature.
The half-life of carbosulfan with both labels was less than 10 minutes in the dry soil. After 8 days the main residue from the phenyl label was carbofuran (54.5% of the TRR), with 3.5% of 3-hydroxycarbofuran, 2.6% of carbosulfan sulfone, and lesser amounts of phenols or oxidized carbamate metabolites of carbosulfan or carbofuran. The predominant residues from the DBA label after 10 minutes were dibutylamine (38.6% of the TRR), carbosulfan (11.4%), N-formyldibutylamine (6.4%) and N-acetyldibutylamine (1.1%). After 8 days the same compounds were detected, but at very low levels (the rest was unidentified). Degradation was substantially slower with the wet soil treated with DBA-labelled carbosulfan. After 48 hours carbosulfan was still 76.2% of the TRR.
The authors concluded that as the results from irradiated and control soils with both labels were so similar exposure to light had very little effect, suggesting that degradation resulted from soil contact rather than the effect of light. The Meeting could not draw such a firm conclusion from the data, although it is likely that the soil was the main contributor to the degradation.
Photolytic and hydrolytic degradation were also investigated in water buffered at pH 7 and distilled water with both DBA- and phenyl-labelled [14C] carbosulfan (5 mg/l) and irradiation for up to 8 days with a sun lamp. Apart from specifying that the radiation was above 300 nm, the spectral characteristics of the sun lamp were not reported nor was the kept. The half-life was about 1.4 days in buffered water and 4-8 days in distilled water. Degradation was much more rapid in irradiated samples than in controls, although the identified products were the same. Degradation was mainly to carbofuran and dibutylamine. Other lesser products from the phenyl label were carbosulfan sulfone, the 7-phenol and the 3-keto-7-phenol. From the DBA label the main product was dibutylamine, with lesser amounts of N-formyldibutylamine and N-acetyldibutylamine.
No other studies on environmental fate were submitted to the Meeting.
Methods of analysis. A number of analytical procedures are available for the determination of carbosulfan, its carbamate and phenolic metabolites and dibutylamine in citrus and animal products. Recent methods used in some of the field trials with citrus fruit and animals are based on the extraction of carbosulfan with dichloromethane (from citrus) or acetone (from animals products) and clean-up on solid-phase extraction (SPE) cartridges before analysis. Carbamate and phenolic metabolites are hydrolysed with HCl before SPE column extraction and dibutylamine is extracted with methanol/water. Some procedures include liquid-liquid partitions.
The HPLC configuration for the determination of carbosulfan includes two post-column reactors, one with H2SO4 to hydrolyse carbosulfan to carbofuran and the other with o-phthalaldehyde + N,N-dimethyl-2-mercaptoethylamine to form a chromophore for fluorescence detection. The configuration is the same for carbamate metabolites, except that only the second reactor is used. Phenolic fractions are derivatized with pentafluorobenzyl bromide (PFBBr), and 3-hydroxy-7-phenols also by ethylation, before analysis. Dibutylyamine fractions are derivatized with dansyl chloride for analysis. Both the phenolic and DBA derivatives are analysed by GC-MS with single ion monitoring.
In citrus a limit of determination of 0.05 mg/kg for all analytes would appear to be supported for this group of methods by adequate recoveries and sample chromatograms, but citrus controls consistently had apparent DBA levels up to 0.02 mg/kg. For this reason 0.1 mg/kg may be a more realistic limit of determination for DBA. For animal products limits of determination of 0.025 mg/kg in milk and 0.05 mg/kg in tissues also appear to be supported for all compounds on the basis of adequate recoveries and sample chromatograms, but DBA was again reported near the limit of determination in some milk samples (0.005-0.037 mg/kg). The methods were independently validated.
The methods used in some other citrus trials involved hexane/propanol extraction of carbosulfan and carbofuran, and HCl reflux extraction of 3-hydroxycarbofuran after ethoxylation, followed by liquid/liquid partition, Florisil column clean-up and GLC with NP detection. The reported limit of determination was 0.01 mg/kg and the methods were validated at that level. However sample chromatograms and corroborating information from multi-residue methods indicated that 0.05 mg/kg would appear to be a more practical limit of determination; it was also recommended as the reporting level.
Older methods used in citrus processing studies were similar to that just described, including GLC with NP detection, but with different clean-up columns. A limit of determination of 0.05 mg/kg is again reasonable, except perhaps for citrus oil where 0.1 mg/kg might be more realistic. Published multi-residue methods were not adequate for carbosulfan, mainly owing to low detector sensitivity.
Stability of residues in stored analytical samples. In a 1980 storage stability study no significant losses of carbosulfan were observed when orange and alfalfa samples fortified with carbosulfan were stored for one year at -18°C. Carbofuran was the predominant metabolite. However, in a pH 4.8 silt loam soil in this same study carbosulfan was almost completely degraded after only three hours at -18°C. This was attributed to the acidity, although carbosulfan was stable in the orange samples, also likely to be acidic. It was more stable in a pH 6 silty clay loam and a pH 6.8 sandy loam at -18°C, with half-lives of about 220 and 144 days respectively.
An interim report described studies of the stability of carbosulfan, its carbamate and phenolic metabolites and DBA in laboratory-fortified oranges and processed orange products stored for up to a year at -18°C. Samples of whole oranges, dried pulp, juice, molasses and oil were fortified at 0.25 mg/kg and most samples were taken for analysis on day 0 and after approximately 3, 6, and 12 months. On day 0 no residues were detected in the juice, and residues in molasses and oil were only 0.08 and 0.12 mg/kg respectively. In these cases carbofuran was the main product of carbosulfan degradation as demonstrated by mass balance investigations. Later samples of juice, molasses and oil were therefore analysed for carbosulfan. Orange oil was analysed for carbosulfan metabolites only on day 0 and after 12 months. There were no appreciable losses of the other carbamate or phenolic residues in any of the samples. It was reported that results of analyses for DBA after 18 and 24 months will be available at an unspecified future time.
The stability and mass balance of residues in cow milk and tissues fortified at 0.25 mg/kg with carbosulfan and DBA were investigated after storage at -18°C for intervals up to 8 months. Carbosulfan was shown to be degraded rapidly in milk, muscle and liver with losses of 16 and 4% from milk and liver respectively in the first month and of 84% from milk, 100% from muscle and 80% from liver after 8 months. Losses of DBA from milk and liver were 8 and 4% respectively after 6 months, but DBA residues in muscle showed an apparent increase of 52% after 6 months. Overall the analyses for DBA were erratic over the test period. Mass balance studies showed that carbosulfan + carbofuran accounted for 68% of the fortification level in milk after 3 months and 88 and 44% in muscle and liver respectively after 6 months. After 8 months carbofuran and the 7-phenol together accounted for over half of the fortification level in milk and liver and 148% in muscle.
These results confirm the instability of carbosulfan per se in animal products even under frozen storage conditions, and add confidence to the prediction that there is little likelihood of finding it in animal products as a result of using carbosulfan on citrus. The results also confirm that degradation is likely to be mainly to carbofuran and its metabolites. The trials did not include analyses for 3-ketocarbofuran, 3-hydroxycarbofuran, or other minor carbamate metabolites. On the basis of the results of the cow feeding study it is likely that 3-hydroxycarbofuran especially may constitute a significant proportion of the residue unaccounted for in these studies.
Citrus residue trials. 30 supervised trials were conducted in 1993-4 in Brazil, Mexico and Spain using the analytical methods described above. The six 1993 Brazilian trials on Valencia and Pera Coroa oranges with a CE formulation (c.1-1.7 g ai/tree) were typical. Six trees/trial were treated, and 4 oranges/tree or 24 oranges/trial sample were taken. Duplicate samples were analysed separately in each trial and both results are tabulated. All trials were according to GAP (a maximum of 2 foliar applications at 0.9-1.7 g ai/tree, the first after full bloom and the 2nd approximately 50 days later). Sprays were to run-off and the 7-day GAP PHI was observed. Sampling, transport and storage were adequate to provide confidence in sample integrity. Analytical results were not corrected for recoveries. At the 7-day GAP PHI the maximum residues were carbosulfan <0.01-0.03 mg/kg, carbofuran 0.02-0.06 mg/kg, and 3-hydroxycarbofuran <0.01-0.03 mg/kg. The 3-ketocarbofuran metabolite occurred in only one trial, at 0.02 mg/kg. Total phenols were up to 0.02 mg/kg and dibutylamine up to 0.15 mg/kg.
A 7-day GAP PHI was also observed in the 7 Mexican trials on Valencia and other oranges with an LE formulation (250 g ai/ha). Residues from application according to GAP, corrected for recoveries, were <0.01-0.08 mg/kg carbosulfan, 0.08-0.26 mg/kg carbofuran, <0.01-0.14 mg/kg 3-hydroxycarbofuran, <0.01-0.04 mg/kg 3-ketocarbofuran, and <0.01-0.14 mg/kg 3-hydroxycarbofuran. Residues of total phenols (uncorrected) were 0.01-0.12 mg/kg and of DBA up to 0.14 mg/kg.
In four 1994 Spanish trials according to GAP with an LE formulation (c.3 g ai/tree) both mature (112-day PHI) and immature (28-day PHI) Valencia oranges were analysed. No residues (<0.01 mg/kg) of carbosulfan were detected at either PHI. In the mature oranges carbofuran residues (corrected for recovery) were <0.01-0.36 mg/kg, 3-ketocarbofuran <0.01-0.05 mg/kg, and 3-hydroxycarbofuran 0.02-0.14 mg/kg. The residues of total phenols (uncorrected) were up to 0.25 mg/kg and of DBA up to 0.14 mg/kg. Not unexpectedly, residues (except of 3-ketocarbofuran) were substantially higher in the immature oranges with a corrected maximum of 0.82 mg/kg carbofuran, <0.01 mg/kg 3-ketocarbofuran, and 0.39 mg/kg 3-hydroxycarbofuran. Maximum (uncorrected) residues of total phenols were 0.63 mg/kg and of DBA 0.29 mg/kg.
Additional Spanish trials according to GAP were conducted in 1993 and 1994 with high-volume applications of an EC formulation (937.5 g ai/ha, 3000 l/ha). Sampling was not only at the harvest GAP PHI (84-147 days), but also at days 0, 30, 45, 60...147. Analyses were only for carbosulfan, carbofuran, and 3-hydroxycarbofuran. Because these compounds are those that the Meeting recommended for inclusion in the definitions of the residues arising from the use of carbosulfan and carbofuran (see below), the results can be used to estimate maximum residue levels and STMRs. The JMPR had previously recommended that all field trials should also include analyses for 3-ketocarbofuran. The Meeting upheld that recommendation with respect to future submissions of data on commodities other than citrus but concluded that, because a number of trials had demonstrated that the compound occurs at relatively low levels in citrus, the data on 3-ketocarbofuran were adequate for citrus fruit.
No residues of carbosulfan were detected at these extended PHIs (84-147 days), but residues of carbosulfan at day 0 (corrected for recoveries) were as high as 3.3 mg/kg. It was seldom detectable after 30 days and even then only in the peel. Carbofuran and especially 3-hydroxycarbofuran were present in some cases after 30 days, and even at harvest after the lengthy PHIs. Generally 3-hydroxycarbofuran was the higher of the two at this stage.
In the six 1993 trials residues at harvest (110-147 days) were <0.01 -0.04 mg/kg carbofuran (mostly <0.01) and 0.05-0.13 mg/kg 3-hydroxycarbofuran. Peel and pulp samples were also analysed at 45 days in two of the trials: carbosulfan was not detected in the peel or pulp (<0.01 mg/kg) and carbofuran residues were about 0.27 or 0.37 mg/kg in the peel and <0.01 mg/kg in the pulp, giving 0.02-0.17 mg/kg in whole oranges. 3-hydroxycarbofuran was at a similar level in the peel and 0.01 mg/kg in the pulp.
In the seven 1994 studies (PHIs 84-140 days) residues in whole oranges were calculated from those in the peel and pulp and the measured peel/pulp weight ratios (24/76, 27/73, 28/72 or 30/70 at harvest, depending on the type of orange). The calculated residues were <0.01 mg/kg carbosulfan, <0.01-0.06 mg/kg carbofuran and 0.04-0.13 mg/kg 3-hydroxycarbofuran. At day 0 the maximum residues in the peel and pulp (uncorrected for recovery) were 0.9 and <0.01 mg/kg carbosulfan, 0.63 and <0.01 mg/kg carbofuran and 0.64 and 0.05 mg/kg 3-hydroxycarbofuran.
The Meeting concluded that MRLs for citrus fruits should be established both for carbosulfan defined as carbosulfan and for carbofuran defined as the sum of carbofuran and 3-hydroxycarbofuran, expressed as carbofuran. The Meeting examined the distribution of data from trials complying with GAP according to these definitions in order to estimate MRLs and STMRs, and observed (not surprisingly) the absence of detectable carbosulfan residues in the Spanish trials with PHIs of 84-147 days compared with the measurable but low levels after 7 days in the Mexican and Brazilian trials. There is much less variation in the sum of carbofuran and 3-hydroxycarbofuran residues however, even with the wide divergence of national PHIs.
Duplicate samples were analysed in most of the trials and the results recorded separately. In order to avoid averaging problems in cases where one of two duplicate results was below the level of detection and to avoid the possibility of over-estimating the median residue, the Meeting decided to treat all the results separately for the estimation of MRLs and STMRs, and included estimated levels for residues between the limits of detection and determination.
The Meeting had two options for the estimation of STMRs for carbosulfan and carbofuran in oranges. One was to use the residues found in the pulp in four 1994 Spanish trials in which carbosulfan was undetected (<0.01 mg/kg) in all four trials and carbofuran + 3-hydroxycarbofuran was undetected in three and at a level of 0.02 mg/kg in the fourth. Since residues were detectable and estimated to be 0.01 mg/kg in each of two 45-day pulp samples in Spanish trials, the residue of 0.02 mg/kg found in the one trial according to GAP is not likely to be aberrant.
The second option was to estimate STMRs for residues of carbosulfan and carbofuran + 3-hydroxycarbofuran in whole oranges from the much larger database of 30 trials. Because of the greater uncertainty associated with the database of only four trials, the Meeting took the second option.
The low or undetectable residues found in the limited number of orange pulp samples and the results of the orange metabolism study described above which showed £ 0.2% and £ 0.3% of the TRR in the pulp and juice respectively give added assurance that residues of carbosulfan and carbofuran + 3-hydroxycarbofuran in the edible portions of oranges, if present, are likely to be very low.
The residues of carbosulfan from all the treatments according to GAP (counting duplicate samples separately) were 0.08, 0.04, 0.03 (3), 0.02 (7), 0.01 (2), and <0.01 mg/kg (39) in a total of 53 samples. If the Spanish trials are excluded, the residues of carbosulfan were 0.08, 0.04, 0.03 (3), 0.02 (7), 0.01 (2), and <0.01 mg/kg (12): 26 samples. From these results, the Meeting estimated a maximum residue level of 0.1 mg/kg and an STMR of 0.01 mg/kg for carbosulfan in oranges. The STMR is at the limit of detection.
The residues of the simple sum of carbofuran + 3-hydroxycarbofuran were 0.5, 0.4, 0.39, 0.33, 0.26, 0.22 (2), 0.19, 0.17 (2), 0.15, 0.14 (2), 0.13 (2), 0.12 (4), 0.11 (6), 0.10, <0.10, 0.09 (3), 0.08 (3), 0.07 (4), <0.07, 0.06 (4), 0.05 (4), 0.04, <0.04, 0.03 (3), and 0.02 (2) mg/kg (53 samples).
On the basis of this distribution, recognizing that the residues would be only very slightly lower if an adjustment were made for the molecular weight of 3-hydroxycarbofuran (about 7% higher than carbofuran), the Meeting estimated a maximum residue level of 0.5 mg/kg and an STMR of 0.1 mg/kg for the sum of carbofuran and 3-hydroxycarbofuran, expressed as carbofuran, in oranges. The Meeting concluded that the STMR for the total carbamate residues would be essentially the same as for carbofuran + 3-hydroxycarbofuran because of the low proportion of carbosulfan in the total carbamate residue.
The Meeting also received information on GAP (without labels) from Thailand for carbosulfan uses on rice, asparagus and watermelons together with what appeared to be an incomplete report of field trials. Although fairly detailed information was provided on the conduct of the trials, no analytical results were included. The information on GAP for these crops was recorded in the evaluation in case the results of the trials become available in the future and provided the GAP is confirmed by approved labels. However, the two trials apparently completed on each of these crops would not be sufficient to estimate maximum residue levels.
Summary information on GAP for German uses on rape, maize and hops was also received but no labels or residue data were provided. Official information on GAP for several commodities was also received from the UK, but again without data on residues. The information on GAP should be re-submitted, together with relevant labels, with any future reports of residue trials.
Feeding studies. Holstein dairy cows were dosed at levels equivalent to 0, 1,3, 10 and 50 ppm carbosulfan in the diet for 28 days. Milk, kidney, liver, muscle and fat were analysed for carbosulfan, carbofuran, 3-hydroxycarbofuran, 3-ketocarbofuran, the 7-phenol, 3-keto-7-phenol and 3-hydroxy-7-phenol metabolites and dibutylamine. Selected cows were held for an additional 3 or 6 days for recovery studies. The residues in the milk and tissues were generally in the decreasing order 3-hydroxycarbofuran, 3-ketocarbofuran, carbofuran and carbosulfan, although in some samples of milk carbosulfan residues were of the same order as those of carbofuran.
No carbamate residues, except one of 7 m g/kg 3-hydroxycarbofuran in the milk of one of three cows at the 10 ppm feeding level after four days, were detected in any samples at the 1, 3 or 10 ppm feeding levels. Phenols were detected at the 10 ppm level, but only in kidney (max. 57 m g/kg 7-phenol and 12 m g/kg 3-hydroxy-7-phenol). Dibutylamine was found at the 10 ppm feeding level up to 54 m g/kg in milk and in all the tissues (highest in kidney at 106 m g/kg). Carbamates were found in the milk and tissues from the 50 ppm feeding level. In summary, the maximum and mean residues at the 50 ppm feeding level were as shown below.
|
Compound |
Maximum/mean residue, m g/kg |
||||||
|
Milk |
Skimmed milk |
Cream |
Kidney |
Liver |
Muscle |
Fat |
|
|
carbosulfan |
12/7 |
ND |
45/28 |
ND |
ND |
ND |
76/44 |
|
carbofuran |
8/4 |
ND |
16/8 |
ND |
ND |
ND |
ND |
|
3-hydroxycarbofuran |
30/19 |
20/10 |
ND |
133/112 |
60/54 |
30/25 |
ND |
|
3-ketocarbofuran |
11/5 |
ND |
ND |
ND |
23/14 |
ND |
ND |
|
7-phenol |
8/4 |
8/6 |
20/11 |
400/358 |
ND |
ND |
14/10 |
|
3-keto-7-phenol |
42/27 |
39/23 |
17/14 |
74/66 |
ND |
ND |
ND |
|
3-hydroxy-7-phenol |
26/19 |
14/12 |
ND |
173/163 |
34/32 |
12/9 |
11/8 |
|
dibutylamine |
119/77 |
- |
- |
890/590 |
294/222 |
58/48 |
47/36 |
where ND = 5 m g/kg in milk and 10 m g/kg in the other substrates, with limits of determination of 25 m g/kg and 50 m g/kg respectively.
At the 50 ppm feeding level carbosulfan was found in milk up to 12 m g/kg, cream up to 45 m g/kg, and fat up to 76 m g/kg, but not in kidney, liver or muscle. At this feeding level carbofuran was found only in milk (up to 8 m g/kg in one cow) and in cream up to 16 m g/kg in a different cow after a 3-day withdrawal period. 3-ketocarbofuran was detected only at the 50 ppm feeding level and then only in the milk and liver at maximum levels (in the same cow) of 11 and 23 m g/kg respectively. No residues were detected in either milk or liver after 3- or 6-day withdrawal periods.
Most of the carbamate residue at the 50 ppm level consisted of 3-hydroxycarbofuran, except in cream and fat. In milk the mean and maximum residues of 3-hydroxycarbofuran were 19 m g/kg after 7 days and 30 m g/kg after 2 days respectively, gradually decreased to 11 m g/kg after 27 days, and were undetectable after a 3-day withdrawal period. There was some reduction of carbosulfan and dibutylamine in fat during the 3- and 6-day recovery periods and no 7-phenol was detected in fat during these periods. The total carbamate residues in milk were fairly constant at approximately 30 m g/kg after the first day of sampling through the dosing period. The 3-hydroxycarbofuran metabolite was up to 133 m g/kg in kidney, £ 60 m g/kg in liver and £ 30 m g/kg in muscle. It was not detected in fat.
Both carbosulfan and carbofuran were found in cream (at mean levels of 28 and 8 m g/kg respectively) but not in skimmed milk (<5 m g/kg). 3-ketocarbofuran was not found in either, and 3-hydroxycarbofuran in skimmed milk (mean level 10 m g/kg) but not in cream, not unexpectedly in view of the polarity afforded by the hydroxyl group.
In the milk, the residues of total phenols were fairly constant over the test period after the first day, with the 3-keto-7-phenol generally predominating. The highest average residues were 3-keto-7-phenol 27 m g/kg, 3-hydroxy-7-phenol 19 m g/kg and 7-phenol 4 m g/kg. These were all undetectable (<5 m g/kg) after 3 or 6 days withdrawal. The highest phenolic residues in the tissues were in the kidney with mean levels of the 7-phenol of 358 m g/kg, the 3-hydroxy-7-phenol of 163 m g/kg and the 3-keto-7-phenol of 66 m g/kg. In the liver and muscle only the 3-hydroxy-7-phenol was detected, at mean levels of 32 and 9 m g/kg respectively, and in fat only the 7-phenol (10 m g/kg) and 3-hydroxy-7-phenol (8 m g/kg).
Apparent dibutylamine was reported in most controls at maximum levels of about 50 m g/kg in both milk and tissue samples, and the residues in treated groups did not correlate well with the dose rates. Its apparent natural occurrence made reliable estimates of the DBA derived from carbosulfan difficult in all samples from animals at the 1 to 10 ppm feeding levels, and in muscle and fat at the 50 ppm level. The mean residues of 590 m g/kg dibutylamine in the kidneys and 222 m g/kg in the livers of the 50 ppm group clearly arose mainly from the treatment however.
Since the highest carbamate residues likely to result from the use of carbosulfan in an animal feed item would be about 2 mg/kg from dry citrus pulp with an STMR of 0.29 mg/kg and this is likely to constitute no more than 20-25% of a cattle diet, and since there were no significant residues at the 10 ppm feeding level and relatively low levels even at 50 ppm, the Meeting concluded that no MRL was required for carbosulfan or its metabolites in milk or tissues to accommodate the use of carbosulfan on citrus. Any residues that might occur would be covered by the maximum residue levels estimated for animal products to accommodate the use of carbofuran (see Section 4.5).
Processing
The Meeting examined reports of two processing studies, one on grapefruit and one on oranges, although data on supervised trials were available only for oranges.
Washing the fruit reduced residues of carbosulfan in grapefruit and oranges by about 67% and 53% respectively. Both carbofuran and total carbamates were reduced by about 21% in grapefruit, but there was no reduction in oranges. The loss of carbosulfan from oranges appears to be offset by increases in carbofuran and 3-hydroxycarbofuran. This situation is analogous to the finding of low or undetectable residues of carbosulfan in harvest samples of oranges although total carbamate residues remain relatively constant over long periods. Because the oranges were processed on the day of the last application instead of after the normal pre-harvest interval, the Meeting decided to consider the total carbamate levels as a measure of the residue in evaluating the processing study. To omit carbosulfan, which would have been largely converted to carbofuran and 3-hydroxycarbofuran at harvest, would underestimate the residues.
No residues (<0.01 mg/kg) of carbosulfan were found in orange juice, so no processing factor could be calculated. Because the residue of 0.17 mg/kg carbosulfan in the whole unwashed oranges is similar to or slightly above the maximum residue found in field trials according to GAP, there would be no real expectation of finding carbosulfan in orange juice. An STMR-P of 0 for carbosulfan per se in orange juice would be reasonable. The residue of 0.73 mg/kg total carbamates is also slightly higher than the residues found from GAP applications in field trials. There was no concentration of any carbosulfan metabolite in the juice, although carbofuran was detected at low levels. A processing factor of about 0.01 for total carbamates applied to an STMR of 0.1 mg/kg for the sum of carbofuran and 3-hydroxycarbofuran would give an STMR-P of 0.001 mg/kg for carbofuran + 3-hydroxycarbofuran in orange juice. Although no MRL for either carbosulfan or the sum of carbofuran and 3-hydroxycarbofuran would appear to be needed since the residues would be expected to be below the limit of detection of 0.01 mg/kg, an MRL of 0.05 mg/kg, at the limit of determination, would be reasonable if one is needed.
The processing factor for carbosulfan on processing unwashed orange fruit to molasses was approximately 0.12. A worst-case STMR-P for carbosulfan in orange molasses would be the STMR for oranges, 0.01 mg/kg, x 0.12 = 0.0012 mg/kg. The processing factor for total carbamate residues was 1.1, and this multiplied by the STMR of 0.1 mg/kg for the sum of carbofuran and 3-hydroxycarbofuran in unwashed whole fruit gives an STMR-P of 0.11 mg/kg. If an MRL for carbosulfan in molasses is needed, a value of 0.05 mg/kg, at the limit of determination, would be appropriate (the maximum expected residue being 0.012 mg/kg). Because there is no significant concentration, the residues of carbofuran + 3-hydroxy carbofuran would not be expected to exceed the maximum residue level of 0.5 mg/kg estimated for whole fruit.
The processing factor for carbosulfan from unwashed oranges to dry pulp was 0.82, so the STMR-P = the STMR for oranges, 0.01 mg/kg, x 0.82 = 0.0082 mg/kg. The STMR-P for the sum of carbofuran and 3-hydroxycarbofuran = STMR 0.1 x processing factor 2.9 = 0.29 mg/kg. On the basis of the 0.82 processing factor and the recommended MRL of 0.1 mg/kg for carbosulfan in whole oranges, 0.1 mg/kg would also be sufficient as an MRL for carbosulfan in dry citrus pulp. The processing factor of 2.9 and the recommended MRL for carbofuran + 3-hydroxy-carbofuran in oranges of 0.5 mg/kg, indicate that 2 mg/kg should be the MRL for the sum of carbofuran and 3-hydroxycarbofuran in dry citrus pulp.
Orange oil. The processing factor for carbosulfan was 7.2 and that for the sum of carbofuran and 3-hydroxycarbofuran was 7.1. Since the STMR levels in oranges were 0.01 and 0.1 mg/kg respectively the corresponding STMR-Ps for orange oil would be 0.072 and 0.71 mg/kg. The same processing factors applied to the recommended MRLs of 0.1 mg/kg for carbosulfan and 0.5 mg/kg for carbofuran in oranges would lead to recommended MRLs of 1 and 5 mg/kg respectively for the oil.
The results of these estimates are summarized below.
|
Sample |
Carbosulfan |
Carbofuran + 3-hydroxycarbofuran |
||||||
|
Processing factor |
Max. res. level1, mg/kg |
Orange STMR, mg/kg |
Processed fraction STMR-P, mg/kg |
Processing factor |
Max. res. level1, mg/kg |
Orange STMR, mg/kg |
Processed fraction STMR-P, mg/kg |
|
|
Whole oranges |
- |
0.1 |
0.01 |
- |
- |
0.5 |
0.1 |
- |
|
Juice |
NF2 |
<0.01 (0.05*) |
0.01 |
0.0 |
0.01 |
0.005 (0.05*) |
0.1 |
0.001 |
|
Molasses |
0.12 |
0.012 (0.05*) |
0.01 |
0.0012 |
1.1 |
0.55 (0.5) |
0.1 |
0.11 |
|
Dry Pulp |
0.82 |
0.08 (0.1) |
0.01 |
0.0082 |
2.9 |
1.5 (2.0) |
0.1 |
0.29 |
|
Oil |
7.2 |
0.72 (1.0) |
0.01 |
0.072 |
7.1 |
3.5 (5.0) |
0.1 |
0.71 |
1 The first number is the estimated maximum residue based on the processing factor and the maximum residue level for whole oranges. The numbers in parentheses are the recommended MRLs. If the estimated maximum residue level is less than the 0.05 mg/kg limit of determination, the limit of determination is recommended as the MRL.2 No factor could be estimated because no residues were detected in the juice
FURTHER WORK OR INFORMATION
Desirable
1. Information on residues of carbosulfan in food in commerce or at consumption2. The final report on the studies of the stability of carbosulfan and its metabolites in oranges and their processed products during frozen storage (final version of Interim Report P-3154)
