Metabolism and environmental fate

The fate of diazinon in animals, plants and soil was described in the 1993 JMPR periodic review or in earlier JMPR evaluations, and only those studies not previously reviewed by the FAO Panel or details of which are needed to facilitate the review and understanding of the studies on animal transfer and ectoparasite control will be described in detail.

Animal metabolism

The fate of diazinon in rats, mice, guinea pigs, dogs, goats, sheep, cows and plants was described in the 1993 periodic review and a diagram of the proposed metabolic pathways was presented. Some of these studies were provided to the WHO Expert Group but not to the FAO Panel. For example, the 1993 Expert Group reviewed several animal disposition or metabolism studies which apparently were not provided to either the 1993 or the present FAO Panel. These included a cow metabolism study (Robins et al., 1957) the disposition of residues in goats (Simoneaux, 1988a) and chickens Simoneaux, 1988b), the identification of metabolites in hens and goats (Simoneaux et al., 1988), a supplementary report on metabolism in hens (Simoneaux et al., 1989), and the characterization of diazinon metabolites in chickens (Simoneaux, 1988c). Some studies reviewed by the 1993 JMPR were re-submitted to the present Meeting.

In mammals

In general terms, diazinon was reported in the 1993 Evaluations (Parts I and II) to be almost completely absorbed from the intestinal tract and easily absorbed dermally. Elimination was reported to be rapid in the urine and faeces, mainly the urine. In mammals, metabolism was reported to progress primarily via hydrolysis of the ester linkage, yielding 4-hydroxy-2-isopropyl-6-methylpyrimidine (metabolite B1 or G-27550) followed by oxidation of the isopropyl group to give primary and tertiary alcohols, of which the latter may become conjugated. Another primary route is oxidation to diazoxon which may be hydrolysed to B 1 or oxidized at the isopropyl group before hydrolysis. Other less important routes include oxidation of the methyl group. Ring cleavage was not reported in rats.

Unchanged diazinon was not a major residue in tissues although low residues of diazinon and diazoxon were reported, especially in fat. Because of the importance of these metabolic routes to the focus of this evaluation on residues in animal products, the proposed metabolic route in mammals presented in the 1993 Evaluations is repeated, slightly modified and expanded, as Figure 1.

In an early study on sheep dosed by stomach tube at 1 g/kg, three cholinesterase-inhibiting metabolites were identified in the urine and fat (Janes et al., 1973). These were hydroxydiazinon (VII in Figure 1), its isomer formed by hydroxylation of the ring methyl group, and dehydro-diazinon, shown in Figure 1 as formed by dehydration of VII. This study was concerned with cholinesterase-inhibiting metabolites, but later studies on metabolism in mammals produced for food have focused on the major routes of metabolism irrespective of cholinesterase inhibition.

Figure 1. Proposed metabolic pathways of diazinon in mammals.

A more recent study (Simoneaux, 1988) was reviewed by the 1993 JMPR (Evaluations Part II - Toxicology). A more detailed description follows. Tissues and milk were analysed after dosing goats by capsule for four consecutive days with [¹⁴C]diazinon at a rate equivalent to 100 ppm in the diet. TLC analysis showed that 94% of the radioactivity was extracted from milk. At least 94% of the total ¹⁴C was generally extractable from the tissues, but only 79% from liver. Over 80% of the extractable ¹⁴C was in the organic phase except that from liver (58%) and kidney (68%). Table 1 indicates the identity and distribution of the residues.

Table 1. Distribution of ¹⁴C in tissues and milk of goats dosed for 4 consecutive days with [¹⁴C]diazinon at a rate equivalent to 100 ppm in the diet (Simoneaux, 1988).

Sample & total 14C expressed as diazinon	% of organoextractable 14C present as
	diazinon	diazoxon	hydroxydiazinon	hydroxypyrimidine	Metab. 31144*
Liver 1.6 mg/kg	0.2	0.3	0.2	19.2	19
Kidney 3.0 mg/kg	<0.1	0.3	<0.1	19.8	30.6
Omental fat 0.4 mg/kg	67.8	4.1	12.8	9.3	6.8
Perirenal fat 0.4 mg/kg	64	0.8	12.3	4.3	4.2
Tenderloin 0.4 mg/kg	6.2	1.0	1.4	26	39.4
Leg muscle 0.5 mg/kg	1.6	<0.1	0.4	35.3	40.4
Milk (day 4) 0.7 mg/kg	0.2	0.2	0.1	39.3	37.3

* Metabolite GS-31144 = hydroxy derivative of hydroxypyrimidine (-OH on tertiary isopropyl carbon, see Figure 1).

The 1993 JMPR also reviewed a study in which residues in tissues were characterized or identified after the dermal treatment of sheep with [¹⁴C]diazinon (Capps and Sumner, 1990). The sheep were treated daily for three days with an acetone solution of [¹⁴C]diazinon at 40 mg/kg bw approximating the maximum drench treatment, applied to a shaved area of c.10% of the back. The sheep were slaughtered 6 hours after the last treatment and tissues were analysed by TLC and HPLC. Over 90% of the ¹⁴C was extracted from all the tissues. The distribution of the residues as determined by HPLC is shown in Table 2. Results were similar by TLC for all samples except muscle, which was not analysed by TLC.

Table 2. Distribution of ¹⁴C residues in sheep tissues after dermal treatment for three days with 40 mg/kg bw [¹⁴C]diazinon as determined by HPLC (Capps and Sumner, 1990)

Sample & total 14C expressed as diazinon1	% of organoextractable 14C present as2
	diazinon	Conj. Of 3114 and hydroxypyrimidine3	Unknown polar compounds	hydroxypyrimidine	Metab. 3114*
Liver 4.4 mg/kg	3.7	13.8	10.9	41.4	18
Kidney 9.4 mg/kg	6.2	8.6	28	24.5	22.6
Back fat 7.3 mg/kg	85.2	-	-	1.6	-
Heart 4.4 mg/kg	55.9	-	-	16.4	12
Leg muscle 4.0 mg/kg	59.2	-	-	23.2	13

* Metabolite GS-31144 = hydroxy derivative of hydroxypyrimidine (-OH on isopropyl tertiary carbon, see Figure 1).

¹ Average of sheep 1 & 2
² Fat, heart and leg muscle from sheep 1. Kidney and liver average of sheep 1 & 2
³ Identified as HPLC "region B", mainly conjugates of GS-3114 and hydroxypyrimidine.

In poultry

Poultry feeding (transfer) studies were provided to the Meeting, but poultry metabolism had not been reviewed by the FAO Panel in the 1993 periodic review. Studies of poultry metabolism were therefore provided on request to the present Meeting (Simoneaux, 1988c, 1989; Simoneaux et al., 1988, 1989).

Simoneaux (1988c) dosed 4 Leghorn hens with [¹⁴C]diazinon by capsule for 7 consecutive days at a rate equivalent to 25 ppm in the diet. Residues were characterized in the excreta, eggs and tissues. More than 78% of the dose was excreted. Simoneaux et al. (1988) identified metabolites in goat urine in order to correlate the findings with residues found in the tissues of goats and hens. The identification of G-27550 and GS-31144 by GC-MS and LC-MS after the acid or enzymatic hydrolysis of aqueous fractions gave evidence of their conjugation.

Simoneaux (1989) and Simoneaux et al. (1989) provided further clarification of hen metabolism, and demonstrated improved extraction of residues and further identification of metabolites after the treatment of samples with protease. The results of analyses of eggs (day 7) and hen tissues by Simoneaux et al. (1989) are summarized in Tables 3-5.

Table 3. Distribution and extractability of ¹⁴C in eggs and tissues before and after treatment with protease (Simoneaux et al., 1989).

Sample	14C		Extractable 14C1 as % of total		% of extractable 14C in
	mg/kg as diazinon	% of dose	Before protease	After protease	Organic phase2	Aqueous phase
Egg yolk	0.07	<0.01	67	88	88	12
Egg white	0.07	0.01	98		87	13
Liver	0.11	0.02	63	82	49	51
Kidney	0.15	0.01	76	98	48	52
Lean meat	0.03	0.05	64	94	49	51
Skin fat	0.02	0.01	44	100	61	39
Peritoneal fat	0.01	0.01	31	100	62	38

¹ Extracted with 9:1 methanol/water
² Methanol/water extract was concentrated and partitioned with hexane

Table 4. Characterization of organo-extractable ¹⁴C in 7-day egg yolks and whites by TLC (Simoneaux et al., 1989).

Residue	Egg yolks		Egg whites
	% of 14C in yolk	mg/kg as diazinon	% of 14C in white	mg/kg as diazinon
Diazinon	0.02	<0.001	0.03	<0.001
Hydroxydiazinon (CGA-14128)	0.06	<0.001	0.05	<0.001
Diazoxon (G-24576)	0.42	<0.001	1.3	<0.001
Pyrimidinol metabolite (G-27550)	11.1	0.007	9.4	0.006
Unknown1	2.9	0.002	-	-
Hydroxy derivative of G-27550 (GS-31144)2	18.6	0.012	33.3	0.022
Metabolite M33 + glucuronide & other conjugates	25	0.016	41.3	0.027

¹ Unresolved GS-31144 and G-27550 suspected
² 4-hydroxy-2-(1-hydroxy-1-methylethyl)-6-methylpyrimidine (Figure 1, structure V).
³ 4-hydroxy-2-(2-hydroxy-1-methylethyl)-6-methylpyrimidine (Figure 1, structure VI).

Similar residues were identified by TLC in organic extracts of poultry tissues (Table 5).

Table 5. Distribution of residues in poultry tissues determined by TLC (Simoneaux et al., 1989).

Residue	% of 14C in sample/residue, mg/kg as diazinon
	Liver	Kidney	Skin	Lean meat	Peritoneal fat
31144	3.5/0.004	3.7/0.006	4.2/0.001	6.5/0.002	3.1/0.001
27550	0.6/0.001	2.3/0.003	2.6/0.001	2/0.001	0.7/0.001
diazoxon	0.9/0.001	0.18/0.001	1.3/0.001	0.24/0.001	0.77/0.001
Unknown1	-----	-----	-----	-----	1/0.001
Unknown2	-----	-----	6.3/0.001	-----	-----
hydroxydiazinon	0.01/0.001	0.11/0.001	0.02/0.001	0.03/0.001	1.4/0.001
diazinon	0.03/0.001	0.080.001	0.89/0.001	0.0.04/0.001	2/0.001
Metabolite M3	2/0.002	5.7/0.008	2.3/0.001	-----	-----
Glucuronide & other conjugates	23.5/0.026.	24.6/0.04	9.7/0.002	-----	-----
Metab. M3 + glucuronide & other conjugates	-----	-----	-----	22.4/0.006	9.7/0.001

¹ Unresolved CGA-14128 suspected
² Unresolved GS-31144 and G-27550 suspected

Plant metabolism

The fate of diazinon in plants was described in the 1993 periodic review and a diagram of the proposed metabolic pathways was presented. The metabolic route in plants is summarized briefly here for convenience. Metabolism in plants progresses, as in animals, primarily by hydrolysis of the ester linkage, yielding metabolite B1 (G-27550), followed by oxidation of the isopropyl group to primary and tertiary alcohols and/or oxidation of the methyl group to the alcohol. Glucose or malonylglucose conjugates are formed from the alcohols. Diazoxon was not reported as a significant plant metabolite although low levels were found in mammals.

The major residues reported in various crops in the 1993 evaluation in decreasing order for each crop are were as follows.

Apple	Beans	Maize forage	Lettuce	Potato foliage1
diazinon/	G-27550	G-27550	G-27550	diazinon
G-27550	GS-31144	JAK-III-57	diazinon	(CL-XIX-293/
	JAK-III-572	GS-31144	GS-31144	GS-31144
	diazinon	diazinon	JAK-III-57	conjugate)
				JAK-III-57
				GS-31144
				G-27550

¹ Diazinon is extensively metabolized in the tuber
² G-27550 with the methyl group oxidized to the alcohol, see 1993 evaluation, Figure 2
³ G-27550 oxidized on the primary carbon of the isopropyl, see 1993 evaluation, Figure 2

Environmental fate in soil

This is described in the 1993 JMPR evaluations.

Environmental fate in water/sediment systems

No information was provided either to the 1993 or the present Meeting.