Plant metabolism

Information was made available to the Meeting on metabolism studies on apples and grapes when thiram was applied to the fruit and leaves, and on soya beans, cotton, wheat and sugar beet when it was used as a seed treatment.

Wyss-Benz(1994) applied [thiocarbonyl-¹⁴C]thiram to the apples and leaves of two apple trees at a dose equivalent to 29.5 kg ai/ha (5 times the label rate) and harvested the fruit and leaves 0, 14, 28, 56 and 101 days after treatment. Surface radioactivity was removed for measurement by washing the fruit or leaves first with acetonitrile and then with acetonitrile/water (9+1). Apples, leaves, peel and pulp were subjected to an exhaustive extraction procedure with acetonitrile and water. Surface-washed apples from days 28 to 101 were homogenized and separated into juice and press cake for extraction and analysis.

The levels and distribution of ¹⁴C on the surface and within the fruit and leaves are recorded in Table 5. Initially, as expected, most of the residue was on the fruit surface, but by day 14 only half of the remaining residue was on the surface. The ¹⁴C incorporated into the fruit (not removed by acetonitrile washing) was quite persistent.

Figure 1. Metabolism of thiram by animals

Table 5. Residues on the surface and within apples and leaves harvested after treating fruit and leaves of two apple trees with [¹⁴C]thiram at a dose equivalent to 29.5 kg ai/ha (Wyss-Benz, 1994). Apple juice and press cake were prepared from acetonitrile-washed apples.

Sample	14C in the apples or leaves expressed as mg thiram/kg and as % of total 14C
	0 days		14 days		28 days		56 days		101 days
	14C as thiram, mg/kg	%	14C as thiram, mg/kg	%	14C as thiram, mg/kg	%	14C as thiram, mg/kg	%	14C as thiram, mg/kg	%
FRUIT
MeCN washings of apples	173	94	10.9	51	0.92	13	0.58	8.0	0.038	0.9
Apple juice					2.1	29	3.2	44	1.98	47
Apple press cake					4.3	58	3.5	48	2.2	53
MeCN-washed apples	11.6	6.3	10.4	49
Apples - TOTAL	185		21		7.3		7.3		4.2
LEAVES
MeCN washings of leaves	3094	82	380	57	125	30	23	8.3	4.9	3.7
MeCN-washed leaves	700	18	292	43	296	70	253	92	128	96
Leaves - TOTAL	3794		672		421		276		133

The distribution of residues between peel and pulp in 5 apples harvested on day 101 was investigated further and is shown right. The major part of the ¹⁴C residue was in the pulp.

Sample	14C as thiram	of total residue
Washings	0.081 mg/kg of whole apples	2.7
Peel	5.2 mg/kg	38
Pulp	1.8 mg/kg	60

The residues in the extracts and acetonitrile washings were characterised by TLC and HPLC. A minor unidentified metabolite, more polar than thiram, was found in the surface washings (Table 6). Thiram was not found within the fruit except on day 0 by TLC at a level estimated to be less than 0.5 mg/kg. The main radioactive residue was very polar. The incorporated residue contained only a small percentage of the dimethyldithiocarbamoyl moiety (Table 7) as demonstrated by the release of small amounts of CS₂ under acid digestion. The ¹⁴C was probably incorporated into natural plant products. The fruit and leaves showed different patterns of metabolites.

Table 6. Occurrence of thiram and unidentified metabolite RO in acetonitrile washings from fruit and leaves after treatment of apple trees with [¹⁴C]thiram at a dose equivalent to 29.5 kg ai/ha (Wyss-Benz, 1994).

Fraction	14C as thiram, mg/kg
	0 days	14 days	28 days	56 days	101 days
FRUIT
Thiram	164	9.5	0.78	<0.3	<0.3
Metabolite RO	7.1	n	n	n	n
Non-resolved 14C	1.8	1.3	0.14	0.58	0.038
LEAVES
Thiram	3008	367	103	n	n
Metabolite RO	86	13	21	n	n
Non-resolved 14C	-	-	-	23	4,9

n: no detectable residues of ¹⁴C (detection limit not stated)

Table 7. CS₂-liberating residues in acetonitrile-washed apples harvested at intervals from apple trees treated with [¹⁴C]thiram at a dose equivalent to 29.5 kg ai/ha (Wyss-Benz, 1994).

Fraction	14C as mg thiram equivalents per kg apple
	0 days	14 days	28 days	56 days	101 days	101 days peel + pulp
Total residue, A	11.6	10.4	6.4	6.7	4.2	2.9
CS2-liberating residue, B	0.104	0.214	0.23	0.22	0.12	0.058
B as % of total of A	0.9	2.1	3.5	3.3	2.8	2.0

Morgenroth and Wyss-Benz (1995) applied [thiocarbonyl-¹⁴C]thiram four times to the grapes and leaves of two grape vines (variety Blauburgunder) at a dose equivalent to 3.2 kg ai/ha (maximum label rate). The growth stages for the 4 applications were 50% petal fall, closure of grapes, change of grape colour and 1 month before maturity. Fruit and leaves were harvested on days 0, 14 and 27 after the final treatment.

Surface radioactivity was removed for measurement by washing the fruit or leaves first with acetonitrile and then with acetonitrile/water (8+2). Surface-washed grapes were homogenized and separated into juice and press cake for extraction and analysis. Washed leaves and fruit components were subjected to an exhaustive extraction procedure with acetonitrile and water.

The total ¹⁴C residues in the grapes and the acetonitrile surface washings are recorded in Table 8. The residues were quite persistent, with approximately one third of the total residues still on the fruit surface 27 days after the final application.

Two unidentified metabolites RO and RI, both more polar than thiram, were detected on the surface of the grapes by HPLC (Table 9), but the levels were substantially lower than those of thiram. Thiram itself constituted most of the surface residue on both fruit and leaves.

Approximately 5% of the residue incorporated within the fruit liberated CS₂ on acid digestion (Table 10), suggesting that it contained the dimethyldithiocarbamoyl moiety.

Most of the ¹⁴C residue in the juice was shown by ultrafiltration to have a molecular weight below 500, and most could not be more positively identified. A glucose conjugate, 1- (N,N-dimethylthiocarbamoylthio)-1-deoxy-b-D-glucose hydrate, was identified at low levels in the juice from grapes harvested 27 days after the final application.

Much of the ¹⁴C in the grapes was very polar or unextractable and had probably become incorporated into natural products.