HANDBOOK OFCHEMICAL RISK ASSESSMENT Health Hazards to Humans, Plants, and Animals ( VOLUME 1 ) - PART 4 doc

Mammals are more resistant than birds to diazinon; the lowestLD50 acute oral value recorded is 224 mg/kg body weight for female rats Rattus rattus.. Table 16.4 Acute Oral Toxicity of Dia

CHAPTER 16 Diazinon

Diazinon, an organophosphorus compound with an anticholinesterase mode of action, wasreleased for experimental evaluation in the early 1950s Diazinon is now used extensively bycommercial and home applicators in a variety of formulations to control flies, cockroaches, lice onsheep, insect pests on ornamental plants and food crops (especially corn, rice, onions, and sweetpotatoes), forage crops such as alfalfa, and nematodes and soil insects in turf, lawns, and croplands(Anonymous 1972; Meier et al 1976; Allison and Hermanutz 1977; Berg 1984; Stone and Gradoni1985; Eisler 1986; Wan 1989; Menconi and Cox 1994; Moore and Waring 1996) Diazinon is themost widely used organophosphorus pesticide in Pakistan to control cabbage root fly and carrotfly (Alam and Maughan 1992) In 1992, more than 612,000 kg diazinon were used in California

on alfalfa, nuts, stone fruits, vegetables, and other crops (Menconi and Cox 1994)

Avian and terrestrial wildlife can acquire diazinon by drinking contaminated water, by absorbing

it through legs and feet, by consuming treated grass or grain, or by ingesting pesticide-impregnatedcarrier particles (Stone and Knoch 1982; Stone and Gradoni 1985) Diazinon was detected at lowconcentrations (<0.2 mg/kg) in tissues of 29% of loggerhead shrikes (Lanius ludovicianus) collected

in Virginia between 1985 and 1988 (Blumton et al 1990) Diazinon poisonings of birds — involving

54 incidents in 17 states — have been recorded for at least 23 species, especially among waterfowlfeeding on recently treated turfgrass Incidents involving agricultural applications may be lessconspicuous, and thus not as well-documented (Stone and Gradoni 1985) Kills of Canada geese(Branta canadensis), brant (Branta bernicla), mallard (Anas platyrhynchos), American black duck(Anas rubripes), American wigeon (Anas americana), other species of waterfowl, and songbirdshave all been associated with consumption of grass or grain shortly after diazinon application(Schobert 1974; Zinkl et al 1978; Stone 1980; Stone and Knoch 1982; Anderson and Glowa 1985;Littrell 1986; Stone and Gradoni 1986; Brehmer and Anderson 1992; Kendall et al 1992, 1993).Fatal diazinon poisonings have also been recorded in humans (Soliman et al 1982; Lox 1983),domestic chickens (Gallus gallus) (Sokkar et al 1975), domestic ducklings (Anas spp.) and goslings(Anser spp.) (Egyed et al 1974, 1976), in laboratory monkey colonies of the tamarin (Saguinus fuscicollis) and the common marmoset (Callithrix jacchus) (Brack and Rothe 1982), and thehoneybee (Apis mellifera) (Anderson and Glowa 1984) Mammals seem to be less sensitive thanbirds to diazinon poisoning (Stone and Gradoni 1985) The lack of reported mammalian mortalities(only one suspected case of a pocket gopher, Thomomys sp., found dead in a park at Yakima,Washington, following aerial spraying of diazinon on shade trees) is consistent with the generalfindings of Grue et al (1983) for organophosphorus insecticides Sublethal effects such as reducedfood consumption and egg production in the ring-necked pheasant (Phasianus colchicus) (Strom-borg 1977), and behavioral modifications, reduced food intake, alterations in liver enzyme activities,

reductions in vitamin concentrations, reduced body temperature, and lowered resistance to coldstress in white-footed mice (Peromyscus leucopus) (Montz and Kirkpatrick 1985) have been noted

at diazinon concentrations markedly lower than those causing acute mortality It has beensuggested — but not proven — that wildlife partially disabled in the field as a result of diazinonpoisoning would be more likely to die of exposure, predation, starvation, or dehydration, or facebehavioral abnormalities, learning impairments, and reproductive declines than would similarlytreated domestic or laboratory animals (Montz 1983; Montz and Kirkpatrick 1985) Sublethal effects

of diazinon on fish populations include vertebral malformations, altered blood chemistry, inhibition

of acetylcholinesterase activity, reduced larval and adult growth, impaired swimming, abnormalpigmentation, histopathology of muscle and gills, and reduction of liver RNA, DNA, and proteincontent (Allison and Hermanutz 1977; Eisler 1986; Moore and Waring 1996)

Diazinon is a broad-spectrum insecticide that is effective against a variety of orchard, vegetable,and soil pests, ectoparasites, flies, lice, and fleas It exists as technical-grade product, wettablepowder, emulsifiable concentrate, granules, and in a variety of other formulations (Negherbon 1959;Anonymous 1972; Eberle 1974; Berg 1984; Menconi and Cox 1994) The active ingredient indiazinon is phosphorothioic acid O,O-diethyl O-(6-methyl-2-1(methylethyl)-4-pyrimidinyl) ester(Figure 16.1) Its molecular formula and molecular weight are C12H2lN2O3PS and 304.35, respec-tively The technical grade is light amber to dark brown and boils at 83° to 84°C Diazinon issoluble in water to 60 mg/L and dissolves readily in aliphatic and aromatic solvents, alcohols, andketones Diazinon can be stored on the shelf for at least 3 years with negligible degradation.Diazinon is also known as G-24480, Sarolex, Spectracide (Anonymous 1972), AG-500, Alfa-tox,Basudin, Dazzel, Diazajet, Diazide, Diazol, ENT 19507, Gardentox, Neocidol, Nucidol, CAS 333-41-5 (Hudson et al 1984), Diagran, Dianon, DiaterrFos, Diazatol, Dizinon, Dyzol, D.z.n., Fezudin,Kayazinon, Kayazol, Knox Out, and Nipsan (Berg 1984)

Some diazinon formulations contain 0.2 to 0.7% (2000 to 7000 mg/kg) of Sulfotep (tetraethyldithiopyrophosphate) as a manufacturing impurity Sulfotep is reportedly at least 100 times moretoxic than diazinon to some organisms (Jarvinen and Tanner 1982) It seems that additional research

is warranted on diazinon/Sulfotep interactions

Diazinon degrades rapidly in plants, with half-time persistence usually less than 14 days.However, persistence increases as temperatures decrease, and is longer in crops with a high oilcontent (Table 16.1) In water, diazinon breaks down to comparatively nontoxic compounds withlittle known hazard potential to aquatic species (Meier et al 1976; Jarvinen and Tanner 1982),although the degradation rate is highly dependent on pH (Table 16.1) The half-time persistence of

Figure 16.1 Structural formula of diazinon.

diazinon on sandy loam soil exposed to sunlight is 2.5 to 10 days (Menconi and Cox 1994) Inmost soils, diazinon seldom penetrates below the top 1.3 cm (Kuhr and Tashiro 1978; Branhamand Wehner 1985) But diazinon may remain biologically available in soils for 6 months or longer

at low temperature, low moisture, high alkalinity, and lack of suitable microbial degraders ymous 1972; Bartsch 1974; Meier et al 1976; Allison and Hermanutz 1977; Menzie 1978; Forrest

(Anon-et al 1981; Branham and Wehner 1985) Bacterial enzymes, derived from Pseudomonas sp., can

be used to hydrolyze diazinon in soil, although costs are prohibitive except in treating emergencysituations involving spills of concentrated diazinon solutions In one case, diazinon was enzymat-ically hydrolyzed within 24 h in an agricultural sandy soil at concentrations as high as 10,000mg/kg (Barick and Munnecke 1982)

In almost every instance of diazinon poisoning, there has been a general reduction in erase activity levels, especially in brain and blood Diazinon exerts its toxicity by binding to theneuronal enzyme acetylcholinesterase (AChE) for a considerable time postexposure (Montz 1983;Kendall et al 1992; Decarie et al 1993) It is emphasized that all organophosphorus pesticidecompounds, in sufficient dose, inhibit AChE in vivo, and all share a common mechanism of acutetoxic action (Murphy 1975) AChE inhibition results in the accumulation of endogenous acetyl-choline in nerve tissues and effector organs, resulting in signs that mimic the muscarinic, nicotinic,and central nervous system (CNS) actions of acetylcholine The immediate cause of death in fatalorganophosphorus compound poisonings, including diazinon, is asphyxia resulting from respiratoryfailure Contributing factors are the muscarinic actions of bronchoconstriction and increased bron-chial secretions, nicotinic actions leading to paralysis of the respiratory muscles, and the CNSaction of depression and paralysis of the respiratory center (Murphy 1975)

cholinest-Diazinon is not a potent inhibitor of cholinesterase and must be converted to its oxygenanalogues (oxons), especially diazoxon (diethyl-2-isopropyl-6-methylpyrimidin-4-yl phosphate)

in vivo before poisoning can occur (Wahla et al 1976) Diazoxon is about 10,000 times moreeffective in reducing cholinesterase activity levels than diazinon (Fog and Asaka 1982) At leasteight diazinon metabolites have been identified in vertebrates, of which four are oxons (Machin

et al 1975; Menzie 1978; Seguchi and Asaka 1981) It is generally agreed that diazinon is olized to diazoxon through the action of liver mixed-function oxidases and nicotinic adeninenucleotide phosphate (Menzie 1978; McLean et al 1984) Diazinon toxicity will depend to someextent on the relation between the rates of activation of diazinon to diazoxon, and of decomposition

metab-of the latter to harmless products (Fujii and Asaka 1982) Birds are more sensitive to diazinon thanmammals, probably because mammalian blood enzymes hydrolyze diazoxon rapidly, whereas birdblood has virtually no hydrolytic activity It seems that diazoxon stability in blood is a major factoraffecting susceptibility of birds and mammals to diazinon poisoning (Machin et al 1975).Diazinon poisoning effects in animals can be delayed or prevented by treatment with a variety

of compounds For example, AChE in diazinon-stressed birds can be reactivated by pralidoxime(Egyed et al 1976; Fleming and Bradbury 1981; Misawa et al 1982) Furthermore, pretreatment

of large white butterfly (Pieris brassicae) larvae with methylene dioxyphenyl compounds willinhibit the diazinon-to-diazoxon activation (Wahla et al 1976) Added tryptophan and its metabo-lites may prevent teratogenic defects by maintaining nicotinic adenine nucleotide (NAD) levels indiazinon-treated chicken embryos; diazinon reportedly acts to decrease the availability of tryptophan

to bird embryos, subsequently interfering with NAD metabolism and causing birth defects erson and Kitos 1982) NAD metabolism in diazinon-stressed birds can also be maintained withnicotinamide (Misawa et al 1982) In contrast to many other organophosphorus insecticides, organ-isms that survive diazinon-inhibited cholinesterase levels can undergo considerable spontaneousreactivation (dephosphorylation), indicating that its dephosphorylation occurs more readily thanthat of cholinesterase inhibited by other organophosphorus compounds (Fleming and Bradbury1981)

(Hend-16.3 LETHAL EFFECTS 16.3.1 General

Diazinon toxicity varies widely within and among species, and is modified by organism age,sex, body size, climatic conditions, pesticide formulation, chemistry of the environment, and otherfactors (Montz 1983) Nevertheless, several trends are apparent, as judged by available data Amongaquatic organisms, for example, freshwater cladocerans and marine shrimps were the most sensitivespecies tested, with LC50 (96 h) values of less than 5 µg/L; freshwater teleosts were more resistant,with the lowest LC50 (96 h) value recorded being 90 µg/L Diazinon has considerable potentialfor causing acute avian poisoning episodes Sensitive species of birds, including ducks, turkey(Meleagris gallopavo), and red-winged blackbird (Agelaius phoeniceus), died at single oral doses

of 2 mg of diazinon/kg body weight Mammals are more resistant than birds to diazinon; the lowestLD50 (acute oral) value recorded is 224 mg/kg body weight for female rats (Rattus rattus) Chronicoral toxicity tests with mammals suggest that daily intake exceeding 5 or 10 mg diazinon/kg bodyweight is probably fatal over time to swine (Sus scrofa) and dogs (Canis familiaris), respectively.Finally, 9 mg/kg of dietary diazinon fed during gestation to pregnant mice (Mus musculus) wasassociated with significant mortality of pups prior to weaning

16.3.2 Aquatic Organisms

Freshwater cladocerans and marine crustaceans were the most sensitive groups tested, withLC50 (96 h) values of less than 2 µg/L for the more sensitive species (Table 16.2) European eels(Anguilla anguilla), rainbow trout (Oncorhynchus mykiss), and bluegills (Lepomis macrochirus)seemed to be the most sensitive freshwater teleosts tested, with LC50 (96 h) values between 80

Table 16.1 Persistence of Diazinon in Plants, Soil, and Water

Sample Type and Other Variables Time for 50% Persistence Reference a

PLANTS

Cabbage leaves

Leafy vegetables, forage crops <2 days 2

Other vegetables, cereal products <7 days 2

a1, Montz 1983; 2, Bartsch 1974; 3, Kuhr and Tashiro 1978; 4, Branham and Wehner

1985; 5, Jarvinen and Tanner 1982; 6, Arthur et al 1983; 7, Meier et al 1976;

8, Allison and Hermanutz 1977; 9, Menconi and Cox 1994.

and 120 µg/L; however, the postlarval and juvenile stages of the striped knifejaw (Oplegnathus fasciatus) — a marine fish cultured intensively in Japan — were unusually sensitive (Table 16.2).

In general, technical grade formulations of diazinon seem to be more toxic than emulsifiableconcentrates, dusts, and oil solutions (Table 16.2) Also, large variations in acute toxicity valueswere evident, even among closely related species (Table 16.2)

Outward signs of diazinon poisoning in fish included lethargy, forward extension of pectoralfins, darkened areas on posterior part of body, hyperexcitability when startled, sudden rapid swim-ming in circles, and severe muscular contractions (Goodman et al 1979; Alam and Maughan 1992).Internally, physiological mechanisms in teleosts preceding death involved the following sequence:cholinesterase inhibition, acetylcholine accumulation, disruption of nerve functions, respiratoryfailure, and asphyxia (Sastry and Sharma 1980) Closely related species of fishes differ markedly

in their sensitivity to diazinon Guppies (Poecilia reticulata) are 5 times more sensitive to diazinonthan are zebrafish (Brachydanio rerio), as judged by LC50 (96 h) values (Keizer et al 1991, 1993).Differences of resistance and accumulation between guppies and zebrafish are related to the rate

of oxidative metabolism Preexposure of guppies to a high sublethal concentration of diazinonincreases resistance to diazinon by a factor of 5 when compared to non-pretreated guppies; zebrafishsimilarly pretreated were not more resistant Pretreatment of guppies resulted in a strong inhibition

of diazoxon formation and pyrimidinol during incubations of diazinon with the hepatic chondrial supernatant It was concluded that toxicity of diazinon in the guppy is due to its metab-olism to a highly toxic metabolite, likely diazoxon And in zebrafish or pretreated guppies havinglow rates of diazinon metabolism, toxicity is due to the accumulation of the parent compound(Keizer et al 1991, 1993) Limited data indicated that the yellowtail (Seriola quinqueradiata), amarine teleost, was 84 times more sensitive to diazinon than were four species of freshwater fishes,

postmito-as judged by LC50 (48 h) values, and by its inability to biotransform diazinon to nontoxic olites within 1 h (Fujii and Asaka 1982) Diazinon has not been detected in marine waters, but thepotential exists for contamination of estuarine areas from agricultural and urban runoff (Goodman

metab-et al 1979)

Table 16.2 Acute Toxicity of Diazinon to Aquatic Organisms (All values

shown are in micrograms of diazinon [active ingredients]

per liter of medium fatal to 50% in 96 h.) Ecosystem, Taxonomic Group, LC50 (96 h)

Organism, and Other Variables ( g/L) Reference a

FRESHWATER

Aquatic Plants >1000 14

Invertebrates

Amphipod, Gammarus fasciatus 0.2 2, 14

Cladoceran, Ceriodaphnia dubia 0.5 14

Daphnid, Daphnia magna

Emulsifiable concentrate (47.5%) 1.3 1

Cladoceran, Simocephalus serrulatus 1.4 b 2

Stonefly, Pteronarcys californica 25 2

Rotifer, Brachionus calyciformes 29,200 14

Fish

European eel, Anguilla anguilla 80 (60–100) 11, 12, 15–17

Rainbow trout, Oncorhynchus mykiss 90–400 2, 3

16.3.3 Birds

Diazinon adversely affects survival of developing mallard embryos when the eggshell surface

is subjected for 30 seconds to concentrations 25 to 34 times higher than recommended field

application rates Mortality patterns were similar for solutions applied in water or in oil (Table 16.3)

Lake trout, Salvelinus namaycush 602 2

Brook trout, Salvelinus fontinalis 770 4

Cutthroat trout, Oncorhynchus clarki 1700 2

Freshwater fish, Barilus vagra 1900–2900 18

Common carp, Cyprinus carpio 3400–5000 18, 19

Fathead minnow, Pimephales promelas 5100–15,000 4, 6

Amphibians

Bullfrog, Rana catesbeiana >2,000,000 c 7

MARINE

Invertebrates

Penaeid shrimp, Penaeus aztecus 28 b 8

Fish

Sheepshead minnow, Cyprinodon variegatus 1470 9

Striped knifejaw, Opelgnathus fasciatus

a1, Meier et al 1976; 2, Johnson and Finley 1980; 3, Anonymous 1972; 4,

Allison and Hermanutz 1977; 5, Sastry and Malik 1982; 6, Jarvinen and Tanner

1982; 7, Hudson et al 1984; 8, Nimmo et al 1981; 9, Goodman et al 1979;

10, Seikai 1982; 11, Sancho et al 1993a; 12, Sancho et al 1992b; 13, Sakr

and Gabr 1992; 14, Menconi and Cox 1994; 15, Ferrando et al 1991; 16,

Sancho et al 1992a; 17, Sancho et al 1993b; 18, Adam and Maugham 1993;

19, Adam and Maugham 1992; 20, Keizer et al 1991.

b 48 h value.

c Single oral dose, in mg/kg body weight.

d 24 h value.

Table 16.2 (continued) Acute Toxicity of Diazinon to Aquatic Organisms

(All values shown are in micrograms of diazinon [active ingredients] per liter of medium fatal to 50% in 96 h.) Ecosystem, Taxonomic Group, LC50 (96 h)

Organism, and Other Variables ( g/L) Reference a

This laboratory finding suggests that eggs of mallards, and probably other birds, are protected when

diazinon is applied according to label directions Chickens dipped in solutions containing 1000 mg

of diazinon/L, an accidentally high formulation, experienced 60% mortality within 3 days; no other

deaths occurred during the next 4 months (Sokkar et al 1975) Results of 5-day feeding trials with

2-week-old Japanese quail (Coturnix japonica), followed by 3 days on untreated feed, showed an

LD50 of 167 mg diazinon/kg diet — a concentration considered “very toxic.” No deaths were

observed at dietary levels of 85 mg diazinon/kg, but 53% died at 170 mg/kg, and 87% at 240 mg/kg

(Hill and Camardese 1986)

Diazinon has a potential for causing acute avian poisoning episodes (Schafer et al 1983)

Ingestion of 5 granules of Diazinon 14G (14.3% diazinon) killed 80% of house sparrows (Passer

domesticus), and all red-winged blackbirds to which they were administered (Balcomb et al 1984)

Ingestion of fewer than 5 granules of Diazinon 14G, each containing about 215 µg diazinon, could

be lethal to sparrow-sized birds (i.e., 15 to 35 g body weight), especially juveniles of seed-eaters

(Hill and Camardese 1984) Acute oral LD50 values indicate that 15 mg diazinon/kg body weight

is fatal to virtually all species tested, and that 2 to 5 mg/kg is lethal to the more sensitive species

(Table 16.4) Signs of diazinon poisoning in birds included muscular incoordination, wing spasms,

wing-drop, hunched back, labored breathing, spasmodic contractions of the anal sphincter, diarrhea,

salivation, lacrimation (tear production), eyelid drooping, prostration, and arching of the neck over

the back (Hudson et al 1984) Most of these signs have been observed in birds poisoned by

compounds other than diazinon; these compounds also act via an anticholinesterase mode of action

(Hudson et al 1984)

Signs of diazinon poisoning in mammals included a reduction in blood and brain cholinesterase

activity, diarrhea, sweating, vomiting, salivation, cyanosis, muscle twitches, convulsions, loss of

reflexes, loss of sphincter control, and coma (Anonymous 1972) Other compounds that produce

their toxic effects by inhibiting AChE, such as organophosphorus pesticides and many carbamates,

show similar effects (Murphy 1975) Two species of marmoset accidentally poisoned by diazinon

exhibited — prior to death — high-pitched voices, trembling, frog-like jumping, a stiff gait, and

pale oral mucous membranes Internally, bone marrow necrosis and hemorrhages in several organs

were evident (Brack and Rothe 1982) Internal damage was also observed in swine and dogs that

died following controlled administration of diazinon Swine showed histopathology of liver and

intestinal tract, and duodenal ulcers; dogs showed occasional rupture of the intestinal wall and

testicular atrophy (Earl et al 1971)

Table 16.3 Mortality of Mallard Embryos after Immersion for 30 seconds in Graded

Strength Diazinon Solutions Age of Eggs Solution Vehicle Diazinon Conc.

Percen

t Approximate Field (days) (water or oil) (mg/L) Dead Application Rate

Modified from Hoffman, D.J and W.C Eastin, Jr 1981 Effects of malathion, diazinon, and

parathion on mallard embryo development and cholinesterase activity Environ Res.

26:472-485.

Results of acute oral toxicity tests indicated that the rat was the most sensitive mammalian

species tested, with an acute oral LD50 of 224 mg diazinon/kg body weight (Table 16.4) It is clear

that mammals are significantly more resistant to acute oral poisoning by diazinon than birds

(Table 16.4) Diazinon was also toxic to mammals when administered dermally, through inhalation,

and in the diet (Table 16.5) The lowest dermal LD50 recorded was 600 mg diazinon/kg body

weight for rabbits (Lepus sp.) using an emulsifiable (4E) formulation The single datum for

inhalation toxicity indicated that 27.2 mg of diazinon/L of air killed 50% of test rabbits after

exposure for 4 h (Table 16.5) Pregnant mice fed diets containing 9 mg of diazinon/kg during

gestation all survived, but some pups died prior to weaning (Table 16.5) Results of chronic oral

toxicity tests of diazinon indicated that death was probable if daily doses exceeded 5 mg/mg body

weight for swine, or 10 mg/kg for dogs (Table 16.5)

Table 16.4 Acute Oral Toxicity of Diazinon to Birds and Mammals (All values

shown are in milligrams of diazinon/kg body weight fatal to 50%

after a single oral dose.) Taxonomic Group, Organism, LD50 (range)

and Other Variables (mg/kg body weight) Reference a

BIRDS

Red-winged blackbird, Agelaius phoeniceus

Mallard, Anas platyrhynchos 3.5 (2.4–5.3) 4, 5

Ring-necked pheasant, Phasianus colchicus 4.3 (3.0–6.2) 4, 5

Northern bobwhite, Colinus virginianus 5.0 b 6

a1, Egyed et al 1974; 2, Schaefer et al 1983; 3, Machin et al 1975; 4, Hudson et al.

1984; 5, Zinkl et al 1978; 6, Hill et al 1984; 7, Anonymous 1972; 8, Earl et al 1971;

9, Wolf and Kendall 1998.

b No mortality seen.

c All animals tested died.

Table 16.5 Toxicity of Diazinon to Laboratory Animals via Dermal, Inhalation, Dietary,

and Chronic Oral Routes of Administration

Mode of Administration, Units,

Organism, Formulations,

and Other Variables Dose Effect Reference a

DERMAL, mg/kg body weight

DIETARY, mg/kg diet, during gestation only

CHRONIC ORAL, mg/kg body weight daily

a1, Anon., 1972; 2, Skinner and Kilgore 1982; 3, Barnett et al 1980; 4, Earl et al 1971.

b Exposure for 4 h to 4% aqueous suspension.

Table 16.4 (continued) Acute Oral Toxicity of Diazinon to Birds and Mammals

(All values shown are in milligrams of diazinon/kg body weight fatal

to 50% after a single oral dose.) Taxonomic Group, Organism, LD50 (range)

and Other Variables (mg/kg body weight) Reference a

with methylene dioxyphenyl compounds antagonized the action of diazinon by a factor of about 2,but synergized the action of diazoxon by an order of magnitude (Wahla et al 1976).

16.4.1 General

Among sensitive species of aquatic organisms, diazinon was associated with reduced growthand reproduction in marine and freshwater invertebrates and teleosts, spinal deformities in fish,reduced emergence in stream insects, measurable accumulations in tissues, increased numbers ofstream macroinvertebrates carried downstream by currents (drift), possible mutagenicity in fish,and interference with algal–invertebrate interactions In birds, diazinon is a known teratogen It isalso associated with reduced egg production, decreased food intake, and loss in body weight.Diazinon fed to pregnant mice resulted in offspring with brain pathology, delayed sexual maturity,and adverse behavioral modifications that became apparent late in life For all groups tested,diazinon directly or indirectly inhibited cholinesterase activity

16.4.2 Aquatic Organisms

Atlantic salmon (Salmo salar) exposed to 0.3 to 45.0 µg diazinon/L for 120 h had reduced

levels of reproductive steroids in blood plasma at all concentrations Exposure to 2 µg/L for only

30 min produced a significant reduction in olfactory response to prostaglandin F2a (Moore andWaring 1996) Carp and other species of freshwater teleosts that survived high sublethal concen-trations of diazinon had impaired swimming and abnormal pigmentation (Alam and Maughan1992) Spinal deformities, mostly lordosis and scoliosis, were among the more insidious effects

documented for diazinon Malformations were observed in fathead minnows (Pimephales promelas)

after 19 weeks in water containing 3.2 µg diazinon/L (Allison and Hermanutz 1977), in yearling

brook trout (Salvelinus fontinalis) within a few weeks at 4.8 µg/L (Allison and Hermanutz 1977),

and in various species of freshwater teleosts after exposure for 7 days to 50 µg diazinon/L

(Kanazawa 1978) Exposure of bluegills (Lepomis macrochirus) to 15 µg diazinon/L for only 24 h

resulted in mild hyperplasia of the gills that increased in severity with increasing concentration(30 to 75 µg/L) and may lead to death (Dutta et al 1993)

Diazinon is a noncarcinogen and reportedly has no significant mutagenic activity in microbialsystems, yeast, and mammals, including humans (as quoted in Vigfusson et al 1983) However,Vigfusson et al (1983) have measured a significant increase in the frequency of sister chromatid

exchange in central mud minnows (Umbra limi) that were exposed in vivo for 11 days to solutions

containing 0.16 to 1.6 µg diazinon/L This finding requires verification

In general, diazinon does not bioconcentrate to a significant degree and is rapidly excreted afterexposure (Menconi and Cox 1994; Tsuda et al 1995) Diazinon in water is bioconcentrated bybrook trout at levels as low as 0.55 µg/L, but tissue residues for all aquatic organisms seldomexceeds 213 times that of ambient water, even after months of continuous exposure (Table 16.6)

Common carp (Cyprinus carpio) exposed to 1.5 to 2.4 µg/L for 168 h had bioconcentration factors

of 12 in muscle, 12 in gallbladder, 50 in kidney, and 51 in liver; almost all was excreted in 72 h

on transfer to clean water, except for kidney, which is the major organ for excretion (Tsuda et al.1990) High bioconcentration factors of 800 in liver, 1600 in muscle, 2300 in gill, and 2730 in

blood are reported for juvenile European eels (Anguilla anguilla) after exposure to 42 to 56 µg/L

for 96 h However, diazinon residues in tissues were usually not detected in tissues after 24 h inclean water (Sancho et al 1992b, 1993a) The half-time persistence of diazinon in tissues ofEuropean eels was estimated at 17 to 31 h in liver, 32 to 33 h in muscle, and 27 to 38 h in gill

Table 16.6 Accumulation of Diazinon by Aquatic Organisms

Ecosystem, Taxonomic Group,

Organism, and Other Variables

Diazinon

Exposure Period a

Topmouth gudgeon, Pseudorasbora parva

a d = days; m = months; pt = posttreatment observation period; LC = life cycle

b1, Kanazawa 1978; 2, Seguchi and Asaka 1981; 3, Allison and Hermanutz 1977; 4, Goodman et al 1979;

5, Tsuda et al 1995.

(Sancho et al 1992a, 1992b, 1993b) Whole guppies exposed to high sublethal concentrations ofdiazinon show bioconcentration factors of 59 after 48 h and 188 after 144 h; the half-time persistence

of diazinon was 10 h after 48-h exposure and 23 h after 144-h exposure (Keizer et al 1993).Diazinon and its metabolites are excreted rapidly posttreatment; the loss rate is approximately linear(Kanazawa 1978) The enzyme system responsible for diazinon metabolism in fish liver microsomesrequired NADPH and oxygen for the oxidative desulfuration of diazinon to diazoxon (Hogan andKnowles 1972) Fish with high fat content contained greater residues of diazinon in fatty tissuesthan did fish with comparatively low lipid content (Seguchi and Asaka 1981), and this could account,

in part, for inter- and intraspecies variations in uptake and depuration Some organisms, such as

the sheepshead minnow (Cyprinodon variegatus), have measurable diazinon residues during initial

exposure to 6.5 µg/L, but no detectable residues after lengthy exposure (Goodman et al 1979),suggesting that physiological adaptation resulting in rapid detoxification is possible

Freshwater and marine alga were unaffected at water diazinon concentrations that were fatal(i.e., 1000 µg/L) to aquatic invertebrates (Stadnyk and Campbell 1971; Shacklock and Croft 1981)

However, diazinon at 1.0 µg/L induced extensive clumping of a freshwater alga (Chlorella dosa) onto the antennae of Daphnia magna within 24 h (Stratton and Corke 1981) The affected

pyrenoi-daphnids were immobilized and settled to the bottom of the test containers The causes of particulatematter adhesion are open to speculation, and additional research is merited

Freshwater macroinvertebrates were comparatively sensitive to diazinon (Table 16.7) Results

of large-scale experimental stream studies (Arthur et al 1983) showed that dose levels of 0.3 µgdiazinon/L caused a five- to eightfold reduction in emergence of mayflies and caddisflies within

3 weeks After 12 weeks, mayflies, damselflies, caddisflies, and amphipods were absent frombenthic samples Elevated (and catastrophic) drift of stream invertebrates was also documented indiazinon-treated streams, especially for amphipods, leeches, and snails (Arthur et al 1983) Short-

term tests of 5-h duration with rotifers (Brachionus calyciflorus) show a 50% reduction in feeding

Table 16.7 Lowest Tested Diazinon Concentrations in Medium that Produced Significant Nonlethal

Biological Effects to Aquatic Organisms

Ecosystem and Taxonomic Group

Concentration ( g/L) Effect Reference a

FRESHWATER

Invertebrates

Fish

Brook trout, Salvelinus fontinalis 0.55 Reduced growth of progeny 3

Fathead minnow, Pimephales promelas 3.2 Reduced hatching success 3

Flagfish, Jordanella floridae 14.0 Reduced larval growth 4

Sheepshead minnow, Cyprinodon variegatus 0.47 Reduced fecundity 3

a1, Arthur et al 1983; 2, Stratton and Corke 1981; 3, Goodman et al 1979; 4, Allison and Hermanutz 1977;

5, Nimmo et al 1981; 6, Dutta et al 1993.

rate on alga (Nannochloris oculata) at 14.2 mg/L (Fernandez-Casalderry et al 1992), with

long-term implications to population stability

Freshwater fish populations can be directly damaged by prolonged exposure to diazinon atconcentrations up to several hundred times lower than those causing acute mortality (Sastry andSharma 1980; Sastry and Malik 1982; Saker and Gabr 1992; Dutta et al 1997; Table 16.7) Impairedreproduction and AChE inhibition occurs concurrently in teleosts during long-term exposure todiazinon, but reproduction can be impaired for at least 3 weeks after fish are placed in uncontam-inated water, even though AChE is normal and they contained no detectable diazinon residues(Goodman et al 1979) Furthermore, diazinon exposure during spawning caused complete, buttemporary, inhibition of reproduction at concentrations that did not produce this effect in fishexposed since hatch (Allison 1977) This could severely impact aquatic species with a shortreproductive period (Allison 1977)

16.4.3 Birds

Diazinon produces visible Type I and II teratisms when injected into chicken embryos (Misawa

et al 1981, 1982; Henderson and Kitos 1982; Wyttenbach and Hwang 1984) Type I teratisms(related to tissue NAD depression) included abnormal beaks, abnormal feathering, and shortenedlimbs Type II teratisms, which included short and wry neck, leg musculature hypoplasia, andrumplessness were associated with disruptions in the nicotinic cholinergic system The severity ofeffects depended on embryo age and was dose related Chick embryos (age 48 h) receiving 25 µg

or more of diazinon/embryo had cervical notochord and neural tube malformations at 96 h, andshort neck at 19 days (Wyttenbach and Hwang 1984) Wry neck occurred at doses ranging from6.2 to 100 µg/embryo, but was more frequent at higher doses Type II teratisms were attributed todisruption of notochord sheath formation Coinjection of 2-pyridinealdoxime methochloride(2-PAM) along with 200 µg diazinon/embryo markedly reduced notochord and neural tube defor-mations (Wyttenbach and Hwang 1984) Similarly, the co-presence of tryptophan — or its metab-olites L-kynurenine, 3-hydroxyanthronilic acid, quinolinic acid — maintained NAD levels of diaz-inon-treated embryos close to, or above, normal, and significantly alleviated the symptoms of Type Iteratisms (Henderson and Kitos 1982)

Reduced egg production, depressed food consumption, and loss in body weight have beenobserved in ring-necked pheasants at daily diazinon intakes greater than 1.05 mg/bird; a dose-related delay in recovery of egg laying was noted after termination of diazinon treatment (Stromborg

1977, 1979) Threshold levels in ring-necked pheasants of 1.05 and 2.1 mg diazinon daily sponded to 1/16 and 1/8 of daily ration (70 g) treated at commercial application rates Foodconsumption of ring-necked pheasants was reduced significantly when only food treated withdiazinon was available; pheasants avoided diazinon-treated food if suitable alternatives existed(Stromborg 1977; Bennett and Prince 1981) Dietary levels above 50 mg/kg were associated withreduced food consumption, weight loss, and reduction in egg production in northern bobwhites(Stromborg 1981) If food reduction is important, then diets containing more than 17.5 mg diazi-non/kg (based on empirical calculations) were potentially harmful to bobwhites (Stromborg 1981).The mechanisms accounting for reduction in egg deposition are not clear, but are probably relatedprimarily to decreased food intake They may also be associated with diazinon-induced pituitaryhypofunction at the level of the hypothalamus, resulting in reduced synthesis and secretion ofgonadotrophic, thyrotrophic, and adrenocorticotrophic hormones (Sokkar et al 1975)

Diazinon exerts its toxic effects by binding to the neuronal enzyme acetylcholinesterase (AChE)for long periods after exposure Diazinon, in turn, is converted to diazoxon, which has a higheraffinity for AChE (and thus greater toxicity) than the parent compound There is a latent period in

white-footed mice in reduction of cholinesterase activities, sometimes up to 6 h, until diazinon isconverted to diazoxon (Montz 1983) Effects of multiple doses of diazinon to mammals are notclear; for example, rats exposed to a high dose of diazinon did not respond fully to a second doseuntil 1 month later (Kikuchi et al 1981) It is difficult to ascertain when complete recovery ofdiazinon-poisoned animals has occurred It is speculated, but not verified, that wildlife recoveringfrom diazinon poisoning may face increased predation, aberrant behavior, learning disabilities,hypothermia, and reproductive impairments (Montz 1983) Data are lacking on recovery aspects

of diazinon-poisoned native mammal populations (Montz and Kirkpatrick 1985)

Diazinon is rapidly biotransformed and excreted in mammals Estimated half-times of diazinonpersistence were 6 to 12 h in rats (Anonymous 1972) and dogs (Iverson et al 1975) Most of the diazinonmetabolites were excreted in the urine as diethyl phosphoric acid and diethyl phosphorothioic acid indogs (Iverson et al 1975), and as hydroxy diazinon and dehydrodiazinon in sheep (Machin et al 1974).Determination of AChE activity in selected tissues following diazinon exposure provided anestimate of potential toxicity, but tissue sensitivity varied widely between and among taxa In sheep,brain cholinesterase inhibition was pronounced after diazinon insult, and metabolism of diazinon

in, or close to, the brain was the most likely source of toxicologically effective diazoxon (Machin

et al 1974, 1975) In rat, diazinon effectively reduced blood cholinesterase levels, with inhibitionsignificantly more evident in erythrocytes than in plasma (Tomokuni and Hasegawa 1985) Allmammalian bloods hydrolyze diazoxon rapidly, whereas birds have virtually no hydrolytic activity

in their blood, and, as a result, were more susceptible than mammals The stability of diazoxon inthe blood appears to be a primary factor in susceptibility to diazinon poisoning (Machin et al.1975) In species lacking blood oxonases, the liver was probably the most important site of diazinonmetabolism (Machin et al 1975) Diazinon that accumulated in rat liver was biotransformed, usuallywithin 24 h, by microsomal mixed-function oxidases and glutathione S-transferases However,diazinon residues in rat kidney were almost 500 times those in liver (and 11 times brain), and weremeasurable in kidney but not in liver (Tomokuni and Hasegawa 1985) It now seems that diazinonresidues in kidney and cholinesterase inhibition in erythrocytes are the most useful indicators ofacute diazinon poisoning in mammals

Sublethal effects of diazinon have been recorded in rodents, the most sensitive mammal grouptested Effects were measured at 0.5 mg diazinon/kg in diets of rats for 5 weeks, at 0.18 mg/kgbody weight administered daily to pregnant mice, and at single doses of 1.8 mg/kg body weightfor rat and 2.3 mg/kg body weight for white-footed mice (Table 16.8) Many variables modifydiazinon-induced responses, including the organism’s sex For example, female rats and dogs weremore sensitive to diazinon than males (Earl et al 1971; Davies and Holub 1980a, 1980b; Kikuchi

et al 1981), but male swine were more sensitive than females (Earl et al 1971)

Behavioral deficits observed in offspring of mice exposed to diazinon during gestation indicatedthat prenatal exposure may produce subtle dysfunctions not apparent until later in life (Spyker andAvery 1977) Pregnant mice given a daily dose of 0.18 or 9 mg diazinon per kg body weightthroughout gestation gave birth to viable, overtly normal, offspring But, pups born to mothers ofthe 9 mg/kg groups grew more slowly than controls and were significantly smaller at 1 month thancontrols (Spyker and Avery 1977) Offspring of mothers receiving 0.18 mg/kg body weight exhibitedsignificant delays in the appearance of the contact placing reflex and in descent of testes or vaginalopening Mature offspring of mothers exposed to either dose level displayed impaired enduranceand coordination on rod cling and inclined plane tests of neuromuscular function (Table 16.8) Inaddition, offspring of the 9-mg/kg-dose group had slower running speeds and less endurance in aswimming test than controls At 101 days, forebrain neuropathology was evident in the 9-mg/kggroup but not in the 0.18-mg/kg group The mechanisms responsible for these effects are unknown(Spyker and Avery 1977)

Diazinon is nonmutagenic to mammals, as judged by its inability to induce sister chromatidexchanges (SCE) in Chinese hamster ovary cells (CHOC) at 80 mg/kg culture medium Mostorganophosphorus insecticides tested induced SCE in CHOC at this concentration (Nishio and

Uyeki 1981; Chen et al 1982) Diazoxon, an oxygen analog of diazinon, did produce SCE at

304 mg/kg, but was 3 to 10 times less effective than oxygen analogues of other organophosphoruscompounds screened (Nishio and Uyeki 1981)

16.4.5 Terrestrial Invertebrates

Tobacco hornworms (Manduca sexta) from a field sprayed with 840 mg diazinon/ha contained

no detectable residues of diazoxon Only one sample, collected about 4 h after spraying, exceeded1.0 mg diazinon/kg body weight No diazinon residues in these insects were detectable after 18 days

Table 16.8 Sublethal Effects of Diazinon in Selected Mammals

Organism and Dose a Exposure Period Effect Referenceb

RAT, Rattus rattus

2 (D) 1 week Depressed plasma cholinesterase (females

only)

3

1000 (D) 3 generations No malformations, no effect on reproduction 5

MOUSE, Mus musculus

(pregnant)

0.18 (BW) 2.8 weeks Altered behavior and delayed sexual maturity

of progeny

6

9 (BW) Throughout gestation Reduced growth and altered serum

immunoglobulins of progeny; some deaths

7

MOUSE (juveniles)

0.18 (BW) 14.4 weeks Impaired endurance and coordination 6

WHITE-FOOTED MICE,

Peromyscus leucopus

2.3 (BW) Single dose 9% depression in brain AChE in 24 h 8 17.3 (BW) Single dose 60% depression in brain AChE in 6 h 9

DOG, Canis familiaris

4 (BW) Single dose 39% reduction in serum cholinesterase in

10 min; 50% reduction in 3.5 h

10

10 (BW) 8 months Testicular atrophy, cholinesterase inhibition 11

SWINE, Sus scrofa

5 (BW) 8 months Cholinesterase inhibition, duodenal ulcers,

4 min Effective lice control for 3 weeks, partial

protection for 8.6 weeks

13 450–650 (BW) Single dose Flesh unfit for human consumption for

several weeks (high fat residues of 333–520 mg/kg)

14

MONKEYS, several

species

a D = mg/kg diet; BW = mg/kg body weight daily.

b1, Davies and Holub 1980a; 2, Kikuchi et al 1981; 3, Davies and Holub 1980b; 4, Lox 1983; 5, Anonymous 1972; 6, Spyker and Avery 1977; 7, Barnett et al 1980; 8, Montz 1983; 9, Montz and Kirkpatrick 1985; 10, Iverson et al 1975; 11, Earl et al 1971; 12, Dressel et al 1982; 13, Wilkinson 1980; 14, Machin et al 1974.

It was concluded that the potential hazard to birds eating hornworms was minimal (Stromborg et al.

1982) In contrast, diazinon residues in molluscan slugs (Agriolimax reticulatus), collected from

plats of spring wheat sprayed with 8000 mg diazinon/ha, increased linearly to about 200 mg/kg at

6 weeks postapplication, then declined to background levels after 16 weeks (Edwards 1976) Duringthis same period, soil residues decreased from about 4 mg/kg immediately after application, toabout 1 mg/kg at 6 weeks, and were not detectable after 12 weeks The high residues observed inslugs may be due, in part, to physical adsorption of diazinon to slug mucus Edwards (1976)concluded that slugs heavily contaminated by diazinon constituted a serious danger to birds andmammals feeding on them

Depuration rates of diazinon differed significantly for two species of nematodes, Panagrellus redivivus and Bursaphelenchus xylophilus (Al-Attar and Knowles 1982) Both species showed

maximum uptake of radiolabeled diazinon between 6 and 12 h, and both metabolized diazinon to

diazoxon and pyrimidinol By 96 h, 95% of the diazinon in P redivivus had been metabolized, but only 26% was transformed in B xylophilus, again demonstrating variability in diazinon metabolism

between related species

As shown earlier, certain aquatic organisms were impacted by diazinon water concentrationsbetween 0.3 and 1.2 µg/L; effects included lowered emergence and elevated drift of stream insects(0.3 µg/L), reduced fecundity of marine minnows (0.47 µg/L), accumulations in freshwater teleosts(0.55 µg/L), and daphnid immobilization (1.0 µg/L) and death (1.2 µg/L) These comparatively lowlevels are of concern because transient peak water concentrations of 4 to 200 µg diazinon/L havebeen recorded near diazinon sheep-dipping sites in England (Moore and Waring 1996), and36.8 µg/L in the Sacramento–San Joaquin River, California (Menconi and Cox 1994) For protection

of sensitive aquatic organisms, Arthur et al (1983) recommended that water diazinon levels shouldnot exceed 0.08 µg/L This value represents a safety factor of about 4 over the lowest recordedadverse effect level of 0.3 µg/L For protection of freshwater aquatic life, Menconi and Cox (1994)recommend an average 4-day concentration of 0.04 µg diazinon/L provided that this value is notexceeded more than once every 3 years and the maximum 1-h concentration does not exceed0.08 µg/L more than once every 3 years Safety factors may require adjustment as additional databecome available Establishment of safe levels is complicated by the fact that diazinon can persistfor many months in neutral or basic waters, including seawater (Kanazawa 1978), but hydrolyzesrapidly in acidic waters (Allison and Hermanutz 1977) Data on chronic effects of fluctuating andintermittent exposures of fishes and invertebrates to diazinon are also needed, and these will aid

in the establishment of safe concentrations for this organophosphorus pesticide (Allison and manutz 1977)

Her-Granular formulations were especially hazardous to seed-eating birds; ingestion of fewer than

5 granules of a Diazinon 14G formulation could be lethal (Hill and Camardese 1984) A reduction

in diazinon content of existing granular formulations may become necessary in application areasfrequented by high densities of seed-eating birds Stone and Gradoni (1985) recommend thatdiazinon should not be used in areas where waterfowl feed, especially turfgrass Suggested alter-

natives to diazinon for turfgrass use include Dursban (O,O-diethyl

O-(3,5,6-trichloro-2-pyridyl)-phosphorothioate), Dylox (dimethyl (2,2,2-trichloro-l-hydroxyethyl) phosphonate), Carbaryl

(1-naphthyl N-methylcarbamate), and Lannate

(S-methyl-N-((methylcarbamoyl)oxy)-thioacetimi-date) (Stone 1980; Stone and Gradoni 1985) Diazinon should be used with caution in large-scalespray applications — such as grasshopper control — as judged by some deaths of horned larks

(Eremophila alpestris), lark buntings (Calamospiza melanocorys), western meadowlarks (Sturnella neglecta), and chestnut-collared longspurs (Calcarius ornatus) when used for this purpose in

Wyoming (McEwen et al 1972) Diazinon applications to agricultural crops comprised a relatively

small percentage of the reported mortality incidents, but it is likely that this category is reported since such incidents were probably less conspicuous than those noted on lawns and golfcourses (Stone and Gradoni 1985) Also, diazinon interactions with other agricultural chemicals,

under-such as Captan (cis-N-((trichloromethyl)thio)-4-cyclohexene-1,2-dicarboximide), may produce

more-than-additive (but reversible) adverse effects on food consumption and egg production ofring-necked pheasants (Stromborg 1977) More research is needed on complex mixtures of agri-cultural pesticides that contain diazinon

In female rats, the no-observable-effect level (NOEL) is 0.1 mg/kg of dietary diazinon At0.5 mg/kg, there was a marked lowering of plasma cholinesterase activity in 5 weeks (Davies andHolub 1980a) But studies with male rats indicate that the NOEL is 2 mg/kg of dietary diazinon,

or about 20 times higher than female rats (Davies and Holub 1980b) Accordingly, future studiesshould consider sex as a variable in toxicity evaluation of diazinon It is generally agreed thatmammals are more resistant than birds to diazinon owing, in part, to their ability to rapidlymetabolize diazoxon However, data are missing on the effects of diazinon on native mammalsunder field conditions, and this should constitute a priority research area No diazinon criteria toprotect human health have been proposed by the U.S Food and Drug Administration or the state

of California (Menconi and Cox 1994)

Diazinon (phosphorothioic acid O,O-diethyl O-(6-methyl-2-(1-methylethyl)-4-pyrimidinyl) ester)

is an organophosphorus compound with an anticholinesterase mode of action It is used extensively

to control flies, lice, insect pests of ornamental plants and food crops, as well as nematodes andsoil insects in lawns and croplands Diazinon degrades rapidly in the environment, with half-timepersistence usually less than 14 days But under conditions of low temperature, low moisture, highalkalinity, and lack of suitable microbial degraders, diazinon may remain biologically active insoils for 6 months or longer

At recommended treatment levels, diazinon-related kills have been noted for songbirds, eybees, and especially waterfowl that consume diazinon-treated grass However, incidents involvingagricultural applications may be underreported Accidental deaths through misapplication of diaz-inon have also been recorded in domestic poultry, monkeys, and humans It has been suggested,but not yet verified, that wildlife partially disabled in the field as a result of diazinon poisoningwould be more likely to die of exposure, predation, starvation, or dehydration, or face behavioralmodifications, learning impairments, and reproductive declines than would similarly treated domes-tic or laboratory animals

hon-Among sensitive aquatic organisms, LC50 (96 h) values of 1.2 to 2.0 µg/L were derived forfreshwater cladocerans, and 4.1 to 5.9 µg/L for marine shrimps; freshwater teleosts were compar-atively resistant, with all LC50 (96 h) values greater than 80 µg/L Sublethal effects were recorded

at 0.3 to 3.2 µg diazinon/L and included reduced emergence of stream insects (0.3 µg/L), reducedfecundity of a marine fish (0.47 µg/L), significant accumulations in freshwater teleosts (0.55 µg/L),daphnid immobilization (1.0 µg/L), potential mutagenicity in a freshwater fish (1.6 µg/L), andspinal deformities in teleosts (3.2 µg/L) Exposure to diazinon during spawning caused temporary,but complete, inhibition of reproduction at concentrations that did not produce this effect in fishexposed continuously since hatch

Acute oral LD50 values of about 2500 to 3500 µg diazinon/kg body weight were determined

for goslings (Anser spp.), ducks (Anas spp.), domestic turkey (Meleagris gallopavo), and the winged blackbird (Agelaius phoeniceus), the most sensitive birds tested A dietary LD50 of 167,000 µg diazinon/kg was determined for Japanese quail (Coturnix japonica) Diazinon produced marked teratogenic effects in embryos of the domestic chicken (Gallus gallus) at 6.2 to

red-25 µg/embryo, reduced egg deposition in the ring-necked pheasant (Phasianus colchicus) at more

than 1050 µg/bird, and (empirically) decreased food consumption and increased weight loss in the

northern bobwhite (Colinus virginianus) at greater than 17,500 µg diazinon/kg diet.

The rat (Rattus rattus) was the most sensitive mammal tested in acute oral toxicity screenings, with an LD50 of 224,000 µg diazinon/kg body weight Chronic oral toxicity tests with swine (Sus scrofa) indicated that death was probable if daily intakes were greater than 5000 µg diazinon/kg body

weight Measurable adverse effects of diazinon have been recorded in rodents, the most sensitivemammalian group tested: at 500 µg/kg in diets fed to rats for 5 weeks, causing blood cholinesterase

inhibition; 180 µg/kg body weight administered daily to pregnant mice (Mus musculus) during

ges-tation, inducing behavioral modifications and delayed sexual maturity of progeny; and single oral

doses of 1800 and 2300 µg/kg body weight in rats and white-footed mice (Peromyscus leucopus),

respectively, producing altered blood chemistry and brain cholinesterase inhibition

For protection of sensitive aquatic organisms, diazinon concentrations in water should notexceed 0.08 µg/L However, more data are needed on effects of fluctuating and intermittent chronicexposures of diazinon on reproduction of fish and aquatic invertebrates Granular formulations ofdiazinon seem to be especially hazardous to seed-eating birds, suggesting a need to control oreliminate granular applications when these species are present For additional protection of birds,diazinon should be used with extreme caution in areas where waterfowl feed, and in large-scalespray applications such as grasshopper control Diazinon in combination with some agriculturalchemicals produced more-than-additive adverse effects on bird growth and fecundity; accordingly,more research is needed on effects of complex mixtures of pesticides that contain diazinon Mostinvestigators agreed that mammals were less susceptible to diazinon than were birds, at least undercontrolled environmental regimens Data are lacking on diazinon impacts to mammals under fieldconditions; acquisition of these data should constitute a priority research area

Alam, M.K and O.E Maughan 1992 The effect of malathion, diazinon, and various concentrations of zinc,

copper, nickel, lead, iron and mercury on fish Biol Trace Elem Res 34:225-236.

Alam, K.K and O.E Maughan 1993 Acute toxicity of selected organophosphorus pesticides to Cyprinus

carpio and Barilus vagra Jour Environ Sci Health B28:81-89.

Al-Attar, H.J and C.O Knowles 1982 Diazinon uptake, metabolism, and elimination by nematodes Arch.

Environ Contam Toxicol 11:669-673.

Allison, D.T 1977 Use of Exposure Units for Estimating Aquatic Toxicity of Organophosphate Pesticides U.S Environ Protection Agen Rep 600/3-77-077 25 pp.

Allison, D.T and R.O Hermanutz 1977 Toxicity of Diazinon to Brook Trout and Fathead Minnows U.S Environ Protection Agen Rep 600/3-77-060 69 pp.

Anderson, J.W and W Glowa 1984 Insecticidal poisoning of honey bees in Connecticut Environ Entomol.

13:70-74.

Anderson, J.F and W Glowa 1985 Diazinon poisoning of brown-headed cowbirds Jour Field Ornithol.

56:407-408.

Anonymous 1972 Diazinon Insecticide Tech Bull., CIBA-GEIGY, Agric Div., Ardsley, NY.

Arthur, J.W., J.A Zischke, K.N Allen, and R.O Hermanutz 1983 Effects of diazinon on macroinvertebrates

and insect emergence in outdoor experimental channels Aquat Toxicol 4:283-301.

Balcomb, R., R Stevens, and C Bowen II 1984 Toxicity of 16 granular insecticides to wild-caught songbirds.

Bull Environ Contam Toxicol 33:302-307.

Barik, S and D.M Munnecke 1982 Enzymatic hydrolysis of concentrated diazinon in soil Bull Environ.

Contam Toxicol 29:235-239.

Barnett, J.B., J.M Spyker-Cranmer, D.L Avery, and A.M Haberman 1980 Immunocompetence over the

lifespan of mice exposed in utero to carbofuran or diazinon: I Changes in serum immunoglobulin concentrations Jour Environ Pathol Toxicol 4(5&6):53-63.

Bartsch, E 1974 Diazinon II Residues in plants, soil, and water Residue Rev 51:37-68.

Bennett, R.S., Jr and H.H Prince 1981 Influence of agricultural pesticides on food preference and

consump-tion by ring-necked pheasants Jour Wildl Manage 45:74-82.

Berg, G.L (ed.) 1984 Farm Chemicals Handbook Meister, Willoughby, OH 501 pp.

Blumton, A.K., J.D Fraser, R.W Young, S Goodbred, S.L Porter, and D.L Luukkonen 1990 Pesticide and

PCB residues for loggerhead shrikes in the Shenandoah Valley, Virginia, 1985–88 Bull Environ Contam.

Toxicol 45:697-702.

Brack, M and H Rothe 1982 Organophosphate poisoning in marmosets Lab Anim 16:186-188.

Branham, B.E and D.J Wehner 1985 The fate of diazinon applied to thatched turf Agron Jour 77:101-104.

Brehmer, P.M and R.K Anderson 1992 Effects of urban pesticide applications on nesting success of

songbirds Bull Environ Contam Toxicol 48:352-359.

Chen, H.H., S.R Sirianni, and C.C Huang 1982 Sister chromatid exchanges in Chinese hamster cells treated

with seventeen organophosphorus compounds in the presence of a metabolic activation system Environ.

Mutagen 4:621-624.

Davies, D.B and B.J Holub 1980a Toxicological evaluation of dietary diazinon in the rat Arch Environ.

Contam Toxicol 9:637-650.

Davies, D.B and B.J Holub 1980b Comparative subacute toxicity of dietary diazinon in the male and female

rat Toxicol Appl Pharmacol 54:359-367.

Decarie, R., J.L DesGranges, C Lepine, and F Morneau 1993 Impact of insecticides on the American robin

(Turdus migratorius) in a suburban environment Environ Pollut 80:231-238.

Dressel, T.D., R.L Goodale, B Zweker, and J.W Borner 1982 The effect of atropine and duct decompression

on the evaluation of diazinon-induced acute canine pancreatitis Ann Surg 195:424-434.

Dutta, H.M., N Qadri, J Ojha, N.K Singh, S Adhikari, J.S.D Munshi, and P.K Ray 1997 Effect of diazinon

on macrophages of bluegill sunfish, Lepomis macrochirus: a cytochemical evaluation Bull Environ.

Contam Toxicol 58:135-141.

Dutta, H.M., C.R Richmonds, and T Zeno 1993 Effects of diazinon on the gills of the bluegill sunfish

Lepomis macrochirus Jour Environ Pathol Toxicol Oncol 12:219-227.

Earl, F.L., B.E Melveger, J.E Reinwall, G.W Bierbower, and J.M Curtis 1971 Diazinon toxicity —

comparative studies in dogs and miniature swine Toxicol Appl Pharmacol 18:285-295.

Eberle, D.O 1974 Diazinon I Analysis of technical grade product, formulations, and residues Residue Rev.

Egyed, M.N., A Shlosberg, M Malkinson, and A Eilat 1976 Some considerations in the treatment of diazinon

poisoned goslings Clin Toxicol 9:245-249.

Eisler, R 1986 Diazinon Hazards to Fish, Wildlife, and Invertebrates: A Synoptic Review U.S Fish Wildl Serv Biol Rep 85 (1.9) 37 pp.

Fernandez-Casalderry, A., M.D Ferrando, and E Andreu-Moliner 1992 Filtration and ingestion rates of

Bra-chionus calyciflorus after exposure to endosulfan and diazinon Comp Biochem Physiol 103C:357-361.

Ferrando, M.D., E Sancho, and E Andreu-Moliner 1991 Comparative acute toxicities of selected pesticides

to Anguilla anguilla Jour Environ Sci Health B26:491-498.

Fleming, W.J and S.P Bradbury 1981 Recovery of cholinesterase activity in mallard ducklings administered

organophosphorus pesticides Jour Toxicol Environ Health 8:885-897.

Forrest, M., K.A Lord, N Walker, and H.C Woodville 1981 The influence of soil treatments on the bacterial

degradation of diazinon and other organophosphorus insecticides Environ Pollut 24A:93-104 Fujii, Y and S Asaka 1982 Metabolism of diazinon and diazoxon in fish liver preparations Bull Environ.

Contam Toxicol 29:453-460.

Goodman, L.R., D.J Hansen, D.L Coppage, J.C Moore, and E Matthews 1979 Diazinon: chronic toxicity

to, and brain acetylcholinesterase inhibition in, the sheepshead minnow, Cyprinodon variegatus Trans.

Amer Fish Soc 108:479-488.

Grue, C.E., W.J Fleming, D.G Bushy, and E.F Hill 1983 Assessing hazards of organophosphate insecticides

to wildlife Trans North Amer Wildl Nat Res Conf 48:200-220.

Henderson, M and P.A Kitos 1982 Do organophosphate insecticides inhibit the conversion of tryptophan

to NAD+ in ovo? Teratology 26:173-181.

Hill, E.F and M.B Camardese 1984 Toxicity of anticholinesterase insecticides to birds: technical grade

versus granular formulations Ecotoxicol Environ Safety 8:551-563.

Hill, E.F and M.B Camardese 1986 Lethal Dietary Toxicities of Environmental Contaminants and Pesticides

to Coturnix U.S Fish Wildl Serv Tech Rep 2 147 pp.

Hill, E.F., M.B Camardese, G.H Heinz, J.W Spann, and A.B DeBevec 1984 Acute toxicity of diazinon is

similar for eight stocks of bobwhite Environ Toxicol Chem 3:61-66.

Hoffman, D.J and W.C Eastin, Jr 1981 Effects of malathion, diazinon, and parathion on mallard embryo

development and cholinesterase activity Environ Res 26:472-485.

Hogan, J.W and C.O Knowles 1972 Metabolism of diazinon by fish liver microsomes Bull Environ Contam.

Toxicol 8:61-64.

Hudson, R.H., R.K Tucker, and M.A Haegele 1984 Handbook of Toxicity of Pesticides to Wildlife U.S.

Fish Wildl Serv Resour Publ 153 90 pp.

Iverson, F., D.L Grant and J Lacroix 1975 Diazinon metabolism in the dog Bull Environ Contam Toxicol.

13:611-618.

Jarvinen, A.W and D.K Tanner 1982 Toxicity of selected controlled release and corresponding unformulated

technical grade pesticides to the fathead minnow Pimephales promelas Environ Pollut 27A:179-195 Johnson, W.W and M.T Finley 1980 Handbook of Acute Toxicity of Chemicals to Fish and Aquatic Inver-

tebrates U.S Fish Wildl Serv Resour Publ 137 98 pp.

Kanazawa, J 1978 Bioconcentration ratio of diazinon by freshwater fish and snail Bull Environ Contam.

Toxicol 20:613-617.

Keizer, J., G D’Agostino, R Nagel, F Gramenzi, and L Vittozzi 1993 Comparative diazinon toxicity in

guppy and zebra fish: different role of oxidative metabolism Environ Toxicol Chem 12:1243-1250.

Keizer, J., G D’Agostino, and L Vittozzi 1991 The importance of biotransformation in the toxicity of

xenobiotics to fish I Toxicity and bioaccumulation of diazinon in guppy (Poecilia reticulata) and zebra fish (Brachydanio rerio) Aquat Toxicol 21:239-254.

Kendall, R.J., L.W Brewer, and R.R Hitchcock 1993 Response of Canada geese to a turf application of

diazinon AG500 Jour Wildl Dis 29:458-464.

Kendall, R.J., L.W Brewer, R.R Hitchcock, and J.R Mayer 1992 American wigeon mortality associated

with turf application of diazinon AG500 Jour Wildl Dis 28:263-267.

Kikuchi, H., Y Suzuki, and Y Hashimoto 1981 Increase of β-glucuronidase activity in the serum of rats

administered organophosphate and carbamate insecticides Jour Toxicol Sci 6:27-35.

Kuhr, R.J and H Tashiro 1978 Distribution and persistence of chlorpyrifos and diazinon applied to turf.

Bull Environ Contam Toxicol 20:652-656.

Littrell, E.E 1986 Mortality of American wigeon on a golf course treated with the organophosphate, diazinon.

Calif Fish Game 72:122-124.

Lox, C.D 1983 Effects of acute pesticide poisoning on blood clotting in the rat Ecotoxicol Environ Safety

7:451-454.

Machin, A.F., P.H Anderson, and C.N Hebert 1974 Residue levels and cholinesterase activities in sheep

poisoned experimentally with diazinon Pestic Sci 5:49-56.

Machin, A.F., H Rogers, A.J Cross, M.P Quick, L.C Howells, and N.F Janes 1975 Metabolic aspects of the toxicology of diazinon I Hepatic metabolism in the sheep, cow, pig, guinea-pig, rat, turkey, chicken

and duck Pestic Sci 6:461-473.

McEwen, L.C., C.E Knittle, and M.L Richmond 1972 Wildlife effects from grasshopper insecticides sprayed

on short-grass range Jour Range Manage 25:188-195.

McLean, S., M Sameshina, T Katayama, J Iwata, C.E Olney, and K.L Simpson 1984 A rapid method to

determine microsomal metabolism of organophosphate pesticides Bull Japan Soc Sci Fish 50:1419-1423.

Meier, E.P., M.C Warner, W.H Dennis, W.F Randall, and T.A Miller 1976 Chemical Degradation of Military Standard Formulations of Organophosphate and Carbamate Pesticides I Chemical Hydrolysis of Diaz- inon U.S Army Med Bioengin Res Dev Lab., Fort Detrick, Frederick, MD Tech Rep 7611 32 pp Menconi, M and C Cox 1994 Hazard Assessment of the Insecticide Diazinon to Aquatic Organisms in the Sacramento–San Joaquin River System Calif Dept Fish Game, Environ Serv Div., Admin Rep 94-2.

60 pp.

Menzie, C.M 1978 Metabolism of Pesticides Update II U.S Fish Wild Serv Spec Sci Rep Wildl No.

212 381 pp.

Misawa, M., J Doull, P.A Kitos, and E.M Uyeki 1981 Teratogenic effects of cholinergic insecticides in chick embryos I Diazinon treatment on acetylcholinesterase and choline acetyltransferase activities.

Toxicol Appl Pharmacol 57:20-29.

Misawa, M., J Doull, and E.M Uyeki 1982 Teratogenic effects of cholinergic insecticides in chick

embryo-T III Development of cartilage and bone Jour Toxicol Environ Health 10:551-563.

Montz, W.E., Jr 1983 Effects of Organophosphate Insecticides on Aspects of Reproduction and Survival in Small Mammals Ph.D thesis Virginia Polytech Inst State Univ., Blacksburg 176 pp.

Montz, W.E., Jr and R.L Kirkpatrick 1985 Temporal patterns of brain cholinesterase activities of

white-footed mice (Peromyscus leucopus) following dosing with diazinon or parathion Arch Environ Contam.

Toxicol 14:19-24.

Moore, A and C.P Waring 1996 Sublethal effects of the pesticide diazinon on olfactory function in mature

male Atlantic salmon parr Jour Fish Biol 48:758-775.

Murphy, S.D 1975 Pesticides Pages 408-453 in L.J Casarett and J Doull (eds.) Toxicology: The Basic

Science of Poisons Macmillan, New York.

Negherbon, W.O 1959 Handbook of Toxicology Vol III Insecticides, A Compendium Natl Acad Sci., Natl.

Res Coun., Div Biol Agric., WADC Tech Rep 55-16:252-261.

Nimmo, D.R., T.L Hamaker, E Matthews, and J.C Moore 1981 An overview of the acute and chronic effects of first and second generation pesticides on an estuarine mysid Pages 3-19 in J Vernberg, A.

Calabrese, F.P Thurberg, and W.B Vernberg (eds.) Biological Monitoring of Marine Pollutants

Aca-demic Press, New York.

Nishio, A and E.M Uyeki 1981 Induction of sister chromatid exchanges in Chinese hamster ovary cells by

organophosphate insecticides and their oxygen analogs Jour Toxicol Environ Health 8:939-946.

Sakr, S.A and S.A Gabr 1992 Ultrastructural changes induced by diazinon and neopybuthrin in skeletal

muscles of Tilapia nilotica Bull Environ Contam Toxicol 48:467-473.

Sancho, E., M.D Ferrando, E Andreu, and M Gamon 1992a Acute toxicity, uptake and clearance of diazinon

by the European eel, Anguilla anguilla (L.) Jour Environ Sci Health B27:209-221.

Sancho, E., M.D Ferrando, E Andreu, and M Gamon 1993a Bioconcentration and excretion of diazinon

by eel Bull Environ Contam Toxicol 50:578-585.

Sancho, E., M.D Ferrando, M Gamon, and E Andreu-Moliner 1992b Organophosphorus diazinon induced

toxicity in the fish Anguilla anguilla L Comp Biochem Physiol 103C:351-356.

Sancho, E., M.D Ferrando, M Gamon, and E Andreu-Moliner 1993b An approach to the diazinon toxicity

in the European eel: bioaccumulation studies Sci Total Environ., Suppl 1993:461-468.

Sastry, K.V and P.V Malik 1982 Acute and chronic effects of diazinon on the activities of three

dehydro-genases in the digestive system of a freshwater teleost fish Channa punctatus Toxicol Lett 10:55-59 Sastry, K.V and K Sharma 1980 Diazinon effect on the activities of brain enzymes from Ophiocephalus (Channa) punctatus Bull Environ Contam Toxicol 24:326-332.

Schafer, E.W., Jr W.A Bowles, Jr., and J Hurlbert 1983 The acute oral toxicity, repellency, and hazard

potential of 998 chemicals to one or more species of wild and domestic birds Arch Environ Contam.

Toxicol 12:355-382.

Schobert, E.E 1974 Ingested insecticide poisoning in waterfowl Jour Zoo Anim Med 5:20.

Seguchi, K and S Asaka 1981 Intake and excretion of diazinon in freshwater fishes Bull Environ Contam.

Toxicol 27:244-249.

Seikai, T 1982 Acute toxicity of organophosphorous insecticides on the developmental stages of eggs, larvae

and juveniles of Japanese striped knifejaw Oplegnathus fasciatus Bull Japan Soc Sci Fish 48:599-603 Shacklock, P.F and G.B Croft 1981 Effect of grazers on Chondrus crispus in culture Aquaculture 22:331-342.

Skinner, C.S and W.W Kilgore 1982 Acute dermal toxicities of various organophosphate insecticides in

mice Jour Toxicol Environ Health 9:491-497.

Sokkar, S.M., M.A Mohammed, and F.A Soliman 1975 Toxic effects of diazinon on the gonads of fowl.

Zbl Vet Med 22A:557-563.

Soliman, S.A., G.W Sovocool, A Curley, N.S Ahmed, S El-Fiki, and A.-K El-Sebae 1982 Two acute

human poisoning cases resulting from exposure to diazinon transformation products in Egypt Arch.

Environ Health 37:207-212.

Spyker, J.M and D.L Avery 1977 Neurobehavioral effects of prenatal exposure to the organophosphate

diazinon in mice Jour Toxicol Environ Health 3:989-1002.

Stadnyk, L and R.S Campbell 1971 Pesticide effect on growth and assimilation in a freshwater alga Bull.

Environ Contam Toxicol 6:1-8.

Stone, W.B 1980 Bird deaths caused by pesticides used on turfgrass N.Y State Turfgrass Conf Proc 4:58-62 Stone, W.B and P.B Gradoni 1985 Wildlife mortality related to use of the pesticide diazinon Northeast.

Environ Sci 4:30-38.

Stone, W.B and P.G Gradoni 1986 Poisoning of birds by cholinesterase inhibitor pesticides Proc Natl.

Wildl Rehab Assn 5:12-28.

Stone, W.B and H Knoch 1982 American brant killed on golf courses by diazinon N.Y Fish Game Jour.

Society for Testing and Materials Philadelphia.

Stromborg, K.L., W.N Beyer, and E Kolbe 1982 Diazinon residues in insects from sprayed tobacco Pages

93-97 in Chemistry in Ecology, Vol 1 Gordon and Breach Science Publishers, Great Britain.

Tomokuni, K and T Hasegawa 1985 Diazinon concentrations and blood cholinesterase activities in rats

exposed to diazinon Toxicol Lett 25:7-10.

Tsuda, T., S Aoki, T Inoue, and M Kojima 1995 Accumulation and excretion of diazinon, fenthion and

fenitrothion by killifish: comparison of individual and mixed pesticides Water Res 29:455-458.

Tsuda, T., S Aoki, M Kojima, and H Harada 1990 Bioconcentration and excretion of diazinon, IBP,

malathion and fenitrothion by carp Comp Biochem Physiol 96C:23-26.

Vigfusson, N.V., E.R Vyse, C.A Pernsteiner, and R.J Dawson 1983 In vivo induction of sister-chromatid exchange in Umbra limi by the insecticides endrin, chlordane, diazinon and guthion Mutat Res 118:61-68.

Wahla, M.A., G Gibbs, and J.B Ford 1976 Diazinon poisoning in large white butterfly larvae and the

influence of sesamex and piperonyl butoxide Pestic Sci 7:367-371.

Wan, M.T 1989 Levels of selected pesticides in farm ditches leading to rivers in the lower mainland of British

Columbia Jour Environ Sci Health B24:183-203.

Wilkinson, F.C 1980 The uptake of dipping fluid and persistence of diazinon on shower-dipped sheep Austral.

Veterin Jour 56:561-562.

Wolfe, M.F., and R.J Kendall 1998 Age-dependent toxicity of diazinon and terbufos in European starlings

(Sturnus vulgaris) and red-winged blackbirds (Agelaius phoeniceus) Environ Toxicol Chem 17:1300-1312.

Wyttenbach, C.R and J.D Hwang 1984 Relationship between insecticide-induced short and wry neck and

cervical defects visible histologically shortly after treatment of chick embryos Jour Exp Zool 229:437-446 Zinkl, J.G., J Rathert, and R.R Hudson 1978 Diazinon poisoning in wild Canada geese Jour Wildl Manage.

42:406-408.

CHAPTER 17 Diflubenzuron

Compounds collectively known as insect growth regulators have been recognized in recentyears as important new insecticides These compounds include juvenile hormone mimics, antiju-venile hormone analogs, and chitin synthesis inhibitors The most widely studied chitin synthesisinhibitor, and the only one currently registered for use against selected insect pests in the UnitedStates, is diflubenzuron (1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea), also known as dimilin(Christiansen 1986; Touart and Rao 1987; Eisler 1992) Chitin is a major component of the toughouter covering, or cuticle, of insects As insects develop from immature larvae to adults, theyundergo several molts, during which new cuticles are formed and old ones are shed Diflubenzuronprevents successful development by inhibiting chitin synthetase, the final enzyme in the pathway

by which chitin is synthesized from glucose (Marx 1977; Ivie 1978)

Diflubenzuron is highly effective against larval stages of many species of nuisance insects Ithas been used extensively to control mosquitoes, midges, gnats, weevils (including the cotton bollweevil, Anthonomus grandis), various beetles, caterpillars of moths and butterflies (especially thegypsy moth, Lymantria dispar), flies, and rust mites (Marx 1977; Ivie 1978; Veech 1978; Schaefer

et al 1980; Opdycke et al 1982a; Muzzarelli 1986) In Maryland, for example, more than 30,000 haare sprayed annually to control gypsy moths (Swift et al 1988a) In general, less than 140 g/ha(2 ounces/acre) of diflubenzuron is sufficient to control susceptible species, although affected larvae

do not die until they molt (Marx 1977)

Most authorities agree that diflubenzuron has low mammalian toxicity, is not highly trated through vertebrate food chains or by absorption from water, remains stable on foliage, andseldom persists for extended periods in soil and water (Marx 1977; Ivie 1978; Schaefer et al 1980).Chitin synthesis inhibitors, however, are not specific to insect pests Beneficial insects also producechitin, as do all arthropods, including spiders, crabs, crayfish, lobsters, shrimp, daphnids, mayflies,stoneflies, barnacles, copepods, and horseshoe crabs All of these groups are adversely affected bydiflubenzuron, including effects on survival, reproduction, development, limb regeneration, andpopulation growth (Farlow 1976; Marx 1977; Christiansen 1986; Cunningham 1986; Muzzarelli1986; Touart and Rao 1987; Weis et al 1987; Eisler 1992; Fischer and Hall 1992)

17.2.1 General

Diflubenzuron breakdown by hydrolysis, soil degradation, or plant and animal metabolisminitially yields 2,6-difluorobenzoic acid and 4-chlorophenylurea Ultimately, the end products are

either conjugated into mostly water-soluble products or are biologically acylated and methylated.

At extremely low doses, diflubenzuron selectively inhibits the ability of arthropods to synthesizechitin at the time of molting, producing death of the organism from rupture of the cuticle or starvation.Other organisms that contain chitin (i.e., some species of fungi and marine diatoms), or polysaccha-rides similar to chitin (i.e., birds and mammals), seem unaffected Mobility and leachability ofdiflubenzuron in soils is low, and residues are usually not detectable after 7 days Degradation ismost rapid when small-particle (2 to 5 µm) formulations are applied and soil bacteria are abundant

In water, diflubenzuron usually persists for only a few days Degradation is most rapid underconditions of high organic and sediment loadings, and elevated water pH and temperature

17.2.2 Chemical and Biochemical Properties

Selected chemical properties of diflubenzuron are listed in Table 17.1 Diflubenzuron tive pathways are almost entirely through cleavage between the carbonyl and amide groups of theurea bridge Ultimately, the end products are either conjugated into predominantly water-solubleproducts or are acylated and methylated biologically (Metcalf et al 1975) Hydrolysis, soil degra-dation, and plant and animal metabolism of diflubenzuron yield the same initial products: 2,6-difluorobenzoic acid and 4-chlorophenylurea Soil degradation and plant and animal metabolisminvolve further conversion of these compounds to 2,6-difluorobenzamide and 4-chloroaniline(Schaefer et al 1980; Gartrell 1981) (Figure 17.1) Interspecies variations in ability to metabolizediflubenzuron are common, as judged by metabolic patterns in rat (Rattus spp.), cow (Bos bovis),and sheep (Ovis aries) In all three species, hydroxylation of either aromatic ring and scission ofthe ureido bridge constituted the main metabolic pathways In cow and rat, the prevailing routewas ring hydroxylation; in sheep, it was the scission reaction In cow and sheep, about half the2,6-difluorobenzoyl moiety excreted in urine was conjugated to glycine, but in rat the acid wasexcreted largely unchanged In sheep, where cleavage-splitting of the diflubenzuron molecule wasthe primary metabolic route, there was no evidence of 4-chlorophenylurea or 4-chloroaniline in

degrada-Table 17.1 Chemical and Other Properties of Diflubenzuron

Chemical names 1-(4-Chlorophenyl)-3-(2,6-difluorobenzoyl)urea; N

-[[(4-chlorophenyl)amino]carbonyl]-2,6-difluorobenzamide); (4-chlorophenyl)urea

1-(2,6-difluorobenzoyl)-3-Alternate names Deflubenzon, Diflubenuron, Dimilin, DU, DU 112307, Duphar BV, ENT-29054,

Largon, Micromite, OMS 1804, PDD 6040-I, PH 60-40, TH 6040, Vigilante Action Insecticide, larvicide, ovicide; insect growth regulator acting by interference with

deposition of insect chitin

Empirical formula C14H9ClF2N2O2Molecular weight 310.68 Formulations Granular; oil-dispersible concentrate; wettable powder Manufacturing process

and impurities

Produced by reaction of 2,6-difluorobenzamide with 4-chlorophenyl isocyanate The technical product is 95% pure Impurities are of low toxicological concern in terminal residues

Stability Stable under sunlight and in neutral or mildly acidic solutions; unstable in strong

basic solutions Physical state White crystalline solid Melting point 210–230°C (technical); 230–232°C (pure) Solubility

Water 0.1–0.2 mg/L at 20°C; 1.0 mg/L at 25°C Polar organic solvents Moderate to good

Data from Metcalf et al 1975; Farlow 1976; Johnson and Finley 1980; Gartrell 1981; Hudson et al 1984; Mayer 1987; Poplyk 1989; Fischer and Hall 1992.

urine (Willems et al 1980) More information on degradation and metabolic pathways of zuron is given in Metcalf et al (1975), Schooley and Quistad (1979), Ivie et al (1980), Willems

difluben-et al (1980), Franklin and Knowles (1981), and Jenkins difluben-et al (1986)

The benzoylphenylureas — including diflubenzuron — control target insect populations atextremely low doses by selectively inhibiting their ability to synthesize chitin-bearing parts Ingesteddiflubenzuron has no apparent adverse effects until the molting process is under way Diflubenzuroncaused increases in cuticle chitinase and cuticle phenoloxidase activity, producing a softenedendocuticle through reduction of its chitin content and a hardened exocuticle as a result of increasedphenoloxidase activity (Farlow 1976) Diflubenzuron inhibits serine protease, thus blocking theconversion of chitin synthetase zymogen into an active enzyme (Cunningham 1986; Muzzarelli1986) Insect larvae treated with diflubenzuron develop cuticles that are unable to withstand theincreased turgor occurring during ecdysis and that fail to provide sufficient muscular support duringmolting These larvae are unable to cast their exuviae, resulting in death from starvation or rupture

of the new, delicate, malformed cuticle (Farlow 1976) In addition to terrestrial insects, zuron is toxic to a wide variety of aquatic insects and crustaceans (Swift et al 1988a, 1988b), but

difluben-it does not seem to affect other organisms that contain chdifluben-itin, including fungi (Muzzarelli 1986)and marine diatoms (Montgomery et al 1990)

Chitin is a polymer of N-acetylglucosamine (AGA), and it rivals cellulose as the most abundantbiopolymer in nature Measured chitin concentrations in marine waters range between 4 and

21 µg/L, and planktonic crustaceans are the most significant source of chitin in the sea (Montgomery

et al 1990) Insect chitin is synthesized during phosphorylation by uridine disphospho-N-acetylglucosamine (UDPAGA) — the immediate precursor of chitin (Crookshank et al 1978) Difluben-zuron inhibits the incorporation of chitin precursors into chitin, with a resultant accumulation ofUDPAGA (Crookshank et al 1978) Chitin is not found in vertebrates, although several importantpolysaccharides similar to chitin are found, including hyaluronic acid (HA) Hyaluronic acid isfound in skin, synovial fluid, connective tissue, vitreous humor, and the covering of the ovum

Figure 17.1 Generalized degradation pattern for diflubenzuron Diflubenzuron (A) degrades initially to

2,6-difluorobenzoic acid (B) and 4-chlorophenylurea (C) Difluorobenzoic acid (B) degrades to difluorobenzamide (D) and 4-chlorophenylurea (C) degrades to 4-chloroaniline (E).

2,6-Hyaluronic acid is a polysaccharide compound of alternating groups of glucuronic acid and AGA.The immediate precursor for glucuronic acid is uridine diphospho-glucuronic acid and that forAGA is UDPAGA Because UDPAGA is used in the synthesis of chitin by insects and of HA byvertebrates, and because diflubenzuron interferes with the incorporation of UDPAGA into chitin

by insects, diflubenzuron may interfere with the formation of HA in birds (Crookshank et al 1978)(to be discussed later)

17.2.3 Persistence in Soil and Water

Mobility and leachability of diflubenzuron in soils is low, and residues are usually not detectableafter 7 days In water, half-time persistence (Tb 1/2) is usually less than 8 days and lowest atelevated temperatures, alkaline pH, and high sediment loadings (Fischer and Hall 1992)(Table 17.2) Increased concentrations of diflubenzuron in soils and waters are associated withincreased application frequency, flooding of treated supratidal areas, wind drift, and excessiverainfall (Cunningham 1986)

Diflubenzuron is persistent in postharvest soils during winter and spring months, especially ifassociated with plant litter Concentrations decline rapidly with the onset of high summer temper-atures to <0.3 mg/kg DW soil in summer (Bull and Ivie 1978; Bull 1980) Diflubenzuron particlesize and soil flora may be important in the soil degradation process Diflubenzuron adsorbed tosmaller particles of 2 µm diameter had a short Tb 1/2 of 3 to 7 days; diflubenzuron adsorbed tolarger particles (10 µm diameter) persisted for 8 to 16 weeks Diflubenzuron adsorbed to particles

of 2 µm diameter had a low rate of degradation in sterile soils (<6% in 4 weeks), but in nonsterilesoils 98% degraded in the same period, suggesting that soil bacteria are important in the degradationprocess (Cunningham 1986) In Canada, data on mobility of a pesticidal chemical in forest soilmust be collected before it can be registered for use under the Canadian Pest Control Products Act

in order to assess its potential for groundwater contamination (Sundaram and Nott 1989) zuron used properly in forest management is unlikely to be leached into groundwater from a site

Difluben-of application (Sundaram and Nott 1989)

Water concentrations of diflubenzuron in treated ponds are significantly higher in surface andmiddle samples than in bottom samples during the first 5 h after treatment However, after 24 h,distribution is about the same for all depths (Colwell and Schaefer 1980) Diflubenzuron persistsfor only a few days in pasture waters at 22 to 45 g/ha applied to control pasture mosquitoes (Aedes nigromaculis, A melanimon); hydrolysis and adsorption onto organic matter limit persistence inwater (Schaefer and Dupras 1977) Aerial spraying of 70 g/ha in a forest ecosystem resulted inpondwater concentrations of 5.9 to 13.8 µg/L, which declined to <0.05 µg/L within 16 to 20 days(Sundaram et al 1991; Tanner and Moffett 1995) Water temperature and pH significantly affectpersistence of diflubenzuron Degradation is most rapid at elevated temperatures and alkaline pHvalues Half-time persistence of diflubenzuron at pH 7.7 and various thermal regimes is 8 days at38°C, 35 days at 24°C, and 29 days at 10°C At pH 10, Tb 1/2 values are 2 days at 38°C, 14 days

at 24°C, and 32 days at 10°C; degradation is negligible at pH 4 and at low temperatures regardless

of pH (Cunningham 1989) In water, as in soil, small-particle (2 to 5 µm diameter) diflubenzuronformulations, such as WP-25%, degrade rapidly, usually in 2 to 8 days (Cunningham 1986) Larger-particle sand-granule formulations, developed for use in mosquito control programs wherein thecompound needs to penetrate thick vegetation to reach the water, reduce drift during applicationand also provide slower release of diflubenzuron into aquatic habitats (Cunningham 1986).The presence of sediments in diflubenzuron marine microcosms results in rapid removal fromseawater and ultimately a reduction in mortality of larval crustaceans (Table 17.2) (Cunningham

et al 1987) But marine sediments that exceed 200 µg diflubenzuron/kg — levels normally tered at application rates for control of salt marsh mosquitoes — could be detrimental to juvenileand adult crustaceans that consume detritus and organic matter on the surface of the marsh or atthe water–sediment interface (Cunningham and Myers 1986; Cunningham et al 1987)

encoun-Table 17.2 Diflubenzuron Persistence in Soil and Water

Sample, Initial Concentration,

2

70, 210, or 630 g/ha applied once

to sandy loam forest soil or clay

loam forest soil, plus water

equivalent to 50.8 cm of

precipitation

Mobility of diflubenzuron was low and did not increase with dosage No residues detected below 10 cm or in leachates in either soil type at all dosage levels At

70 g/ha, all residues were found in the top 2.5 cm; at

630 g/ha, 4–9% moved below 2.5 cm in sandy loam (mobility was lower in clay loam)

3

DISTILLED WATER

100 µg/L, 37°C No degradation at pH 4 in 8 weeks; Tb 1/2 was about

7 days at pH 6 and <3 days at pH 10 Major degradation products were 4-chlorophenylurea and

2,6-difluorobenzoic acid; small amounts of 2,6-difluorobenzamide and a quinazolinedione product were also formed

Maximum concentrations, in µg/L, were 8.8 in 1 h, 7.1 in

24 h, 3.9 in 48 h, and 2.6 in 72 h; most treatments produced ND (<1 µg/L) residues in 24 h

4-chloroaniline increased from 0.7 at 1 h to 2.6 at 4 days

5 µg/L Concentration immediately after treatment was 4.6 µg/L;

after 2 weeks, it was 0.3 µg/L

7

10 µg/L Initial concentration in medium declined from 9.8 µg/L to

0.2 µg/L after 2 weeks

7 13.8 µg/L Concentration 1 h after aerial spraying of 70 g/ha; this

In Europe and elsewhere, diflubenzuron is used in a variety of ways not permitted in the UnitedStates For example, diflubenzuron and other insect growth regulators are fed as admixtures torations of chickens, cattle, and swine in order to control fly larvae breeding in their manures, andalso as a spray directly on manures prior to disposal (Opdycke et al 1982b: Opdycke and Menzer1984; Giga 1987) Diflubenzuron has been administered orally as a bolus to beef cattle for control

of face flies (Musca autumnalis) and horn flies (Haematobia irritans), two serious pests of cattle

in North America Immature insects develop in fresh manure on open pasture A single bolusreleased diflubenzuron into feces that killed horn and face fly larvae for 8 weeks and remainedpartially effective for 16 weeks (Scott et al 1986)

Three diflubenzuron formulations are now in general use: an oil dispersible concentrate, awettable powder (WP), and granules (Bull 1980; Cunningham 1986; Poplyk 1989) Granularformulations are produced by applying diflubenzuron to sand granules Since technical difluben-zuron (99.5% pure) is a crystalline material that is almost insoluble in water (i.e., 0.1 mg/L at20°C), it is usually dispersed in an organic solvent carrier Wettable powders (25% active ingredi-ents), however, are dispersed in water for use in many commercial applications; diflubenzuronparticle size in WP-25 formulations usually ranges between 2 and 5 microns

17.4.1 General

Diflubenzuron applied to foliage of terrestrial plants tends to remain adsorbed for several weekswith little or no absorption or translocation from plant surfaces; loss is mainly by wind abrasion,rain washing, or shedding of senescent leaves Among insect species, there is great variability insensitivity to diflubenzuron In general, diflubenzuron is toxic to early life stages of insects atconcentrations as low as 0.1 mg/kg diet and at topical applications between 0.003 and0.034 µg/larvae Among aquatic organisms, early developmental stages of crustaceans and insectsare the most sensitive groups tested Adverse effects on growth, survival, reproduction, and behavior

100 µg/L Tb 1/2 of 7.9 days at 38°C, and 35 days at 24°C 9

a1, Booth and Ferrell 1977; 2, Schaefer and Dupras 1977; 3, Sundaram and Nott 1989; 4, Ivie et al 1980;

5, Schaefer and Dupras 1976; 6, Schaefer et al 1980; 7, Apperson et al 1978; 8, Cunningham et al 1987;

9, Cunningham and Myers 1986; 10, Sundaram et al 1991.

Table 17.2 (continued) Diflubenzuron Persistence in Soil and Water

Sample, Initial Concentration,

occur between 0.062 and 2.0 µg/L Groups highly resistant to diflubenzuron include the algae,gastropods, fishes, and amphibians Birds are comparatively resistant: acute oral LD50 valuesexceed 2000 mg diflubenzuron/kg body weight (BW), and dietary levels of 4640 mg/kg ration aretolerated for 8 days Also, forest birds seem unharmed by recommended diflubenzuron applicationprocedures to control pestiferous insects No data are available on mammalian wildlife However,studies with small laboratory animals and domestic livestock suggest a high degree of resistance.

No observable adverse effects occur in cows given 0.25 mg/kg BW daily for 4 months, in rabbitsgiven 4 mg/kg BW daily on days 6 to 18 of gestation, in dogs fed diets containing 40 mg/kg for

13 weeks (equivalent to 1.6 mg/kg BW daily), in rats fed diets containing 160 mg/kg for 2 years,and in rabbits and rodents given single oral or dermal doses <2000 mg/kg BW All of these pointsare discussed later

17.4.2 Terrestrial Plants

There is little to no absorption and translocation of diflubenzuron residues from plant surfaces(Gartrell 1981) Due to its stability and low volatility, diflubenzuron residues adhering to plantsurfaces are removed primarily through physical effects such as wind abrasion, rain washing, orthe loss of dead leaves (Bull 1980) A greenhouse study with corn (Zea mays), soybeans (Glycine max), cabbage (Brassica oleracea capitata), and apples (Malus sp.) showed no significant degra-dation of diflubenzuron residues in leaves for up to 16 weeks after treatment (Gartrell 1981) In astudy with radiolabeled diflubenzuron, a single dose applied to a cotton (Gossypium hirsutum) leafshowed <5% photodegradation in 4 weeks, <7% absorption in 7 weeks, <50% loss to weathering

or volatilization in 4 weeks in samples not exposed to rain, and 77% loss in 3 weeks after a heavyrainfall (Bull and Ivie 1978; Bull 1980) Edible portions of rotational crops treated repeatedly withdiflubenzuron at recommended application levels had low, but detectable, residues Maximumconcentrations, in mg/kg DW, were always <0.01 in wheat (Triticum spp.), <0.02 in cotton, <0.09

in collards (Brassica spp.), and <0.16 in radish (Raphanus spp.) (Bull and Ivie 1978; Bull 1980).Diflubenzuron applied aerially to forest leaves in the spring growing season did not persistwhen the leaves were placed in stream water (Harrahy et al 1993) Residues on oak leaves decreased36% in July, and 23% in August within the first 48 h of stream incubation, reaching >90% losswithin 3 weeks However, in December, after 54 days in the stream, there was no significant lossfrom leaves of red maple, oak, or poplar The persistence of diflubenzuron to forest leaves underwinter conditions is attributed to the increased stability of diflubenzuron at cold temperatures, thegreater retention to leaves at low temperatures, and the reduction in microbial degradation In view

of the persistence of diflubenzuron on hardwood leaves at low stream temperatures, nontargetaquatic organisms that consume these leaves may be exposed for extended periods with possibleadverse effects (Harrahy et al 1993)

Foliage of cotton that initially contained 100 mg/kg DW contained about 60 mg/kg after

7 weeks; leaf residues consisted entirely of the parent diflubenzuron (Gartrell 1981) Diflubenzuronapplied topically to lima bean (Phaseolus lunatus) foliage was not absorbed by the plant, asexpected Injected diflubenzuron, however, was metabolized, and certain metabolites were similar

to those isolated from mites (Franklin and Knowles 1981)

Diflubenzuron mixed into compost layers of the cultivated mushroom (Agaricus bisporus) at

30 mg/kg compost to control dipteran pests of mushroom resulted in increased yield and size.However, at higher concentrations of 180 mg/kg and 1080 mg/kg, mushroom yield and numberwere reduced, and this became more severe over time (White 1986) Frequent applications ofdiflubenzuron to agricultural soils are not detrimental to nitrogen-fixing bacteria (i.e., Azotobacter vinelandii), and high concentrations could stimulate nitrogenase activity in soils This conclusion

is based on a study by Martinez-Toledo et al (1988) using nonsterile agricultural soils and sterilizedsoils inoculated with A vinelandii At diflubenzuron loadings between 100 and 500 mg/kg, allconcentrations tested had a stimulatory effect on nitrogen fixation in both soils

17.4.3 Terrestrial Invertebrates

Diflubenzuron is most toxic to early life stages of some insects at 0.1 mg/kg diet, 0.034 µg/larvae(about 3.1 mg/kg BW), or in combination with various chemicals (Table 17.3) Some beneficialinsects, such as the honey bee (Apis mellifera), are adversely affected at dietary concentrations of

1 mg/kg for 12 weeks, 10 mg/kg for 10 weeks, or 59 mg/kg for 10 days (Table 17.3) At 28 to

56 g/ha (0.025 to 0.05 pounds/acre), diflubenzuron effectively controls mosquitoes for 8 to 15 days(Schaefer and Dupras 1977; Booth and Ferrell 1977), especially organophosphorus insecticide-resistant strains of salt marsh mosquitoes in California (Lee and Scott 1989) Diflubenzuron wasalso effective in controlling strains of house fly (Musca domestica) that were resistant to orga-nochlorine, organophosphorus, carbamate, and pyrethroid insecticides on a United Kingdom pigfarm; 416 mg/m2 to slurry pots of pig weaning rooms gave effective control 2 to 4 weeks afterapplication (Webb and Wildey 1986)

Chemical control of larvae of gypsy moth and other forest-insect defoliators may cause criminate reduction of nontarget arthropods, which, in turn, may affect food resources of forestbirds and small mammals This problem is of special concern in West Virginia, where two species

indis-of endangered bats (Indiana bat, Myotis sodalis; eastern big-eared bat, Plecotus phyllotis) occur inareas threatened by gypsy moth defoliation (Martinat et al 1988) Diflubenzuron applications,usually at 70 g/ha on two consecutive days, controlled gypsy moth larvae and also significantlyreduced populations of canopy macrolepidoptera and nonlepidopteran mandibulate herbivores.Sucking herbivorous insects, microlepidoptera, and predaceous arthropods, however, were relativelyunaffected, which suggests that although diflubenzuron can potentially affect food supply of forestbirds and small mammals, these effects are probably minimal (Martinat et al 1988)

Researchers generally agree that diflubenzuron causes incomplete ecdysis by interfering withchitin synthesis However, diflubenzuron at lethal concentrations causes an effect in chironomidlarvae (Chironomus decorus, Tanypus grodhausi) other than inhibition of chitin synthetase, asjudged by histopathology of the alimentary canal, especially the ventriculus Dysfunction of theventriculus, an organ that normally lacks chitin, results in a general breakdown of the digestiveapparatus of exposed chironomid larvae (Pelsue 1985) Exposure of nematodes and of adults ofseveral insect species, including boll weevil, housefly, and stable fly (Stomoxys calcitrans), todiflubenzuron results in deposition of eggs that appear normal but fail to hatch This effect seems

to be due to an ovicidal action and not to sterility of the treated adults, since the larvae appear toundergo normal development within the egg Secretion of unmetabolized diflubenzuron into theeggs apparently accounts for observed ovicidal effects (Ivie and Wright 1978; Veech 1978; Ivie

et al 1980) Treated female boll weevils began to lay viable eggs 12 days after treatment andbecame as productive as controls in 24 days; additional treatment is required to maintain a significantsuppression of egg hatch (Bull 1980)

Diflubenzuron is the most investigated benzoylphenylurea and has shown excellent potency forcontrolling mosquitoes and certain lepidopterous and coleopterous pests Some insect species,however, cannot be controlled efficiently by diflubenzuron For example, the cotton leafworm(Spodoptera littoralis) is comparatively resistant because of reduced penetration through the exo-skeleton, rapid elimination of unchanged diflubenzuron, and rapid metabolism, which occurs mainlythrough hydrolysis (El Saidy et al 1989) To combat Spodoptera and other resistant pests, newbenzoylphenylurea compounds have been developed, including chlorfluzuaron, teflubenzuron, andhexafluron (El Saidy et al 1989)

Beneficial insects associated with fruit orchards show different responses to diflubenzurontreatment (Broadbent and Pree 1984) Lacewings (Chrysopa oculata) in contact with leaves con-taining 300 mg/kg DW had reduced survival and inhibited molting of first instar larvae, but theassassin bug (Acholla multispinosa) was not affected by contact with treated leaves Lacewingsand other beneficial predator insects fed diflubenzuron-treated, two-spotted spider mites (Tetrany- chus urticae) for 3 days showed no adverse effects after 14 days (Broadbent and Pree 1984)

Spraying of diflubenzuron at 28 g/ha to control gypsy moth did not affect Cotesia melanoscela, a

hymenopterid predator of the gypsy moth However, another natural enemy, a virus, was adversely

affected (Webb et al 1989) Certain arthropod predators were unaffected by diflubenzuron at

70 mg/ha applied four times in 3 weeks to control the boll weevil; these include the convergent

lady bug beetle (Hippodamia convergens), the big-eyed bug (Geocoris punctipes), and various

species of Coleomegilla, Orius, Nabis, and Chrysopa (Deakle and Bradley 1982)

Diflubenzuron can be either hydrolyzed at the urea bridge or oxidized by ring hydroxylation

followed by conjugation Hydrolytic cleavage seems to be a major route for diflubenzuron

metab-olism in many insect species (El Saidy et al 1989) Two-spotted spider mites showed <10%

absorption in 96 h of topically applied diflubenzuron Of the amount absorbed, about 27% was

metabolized in 96 h to 4-chlorophenylurea, 2,6-difluorobenzoic acid, 4′-chloroformanilide,

2,6-difluorobenzamide, and other metabolites (Franklin and Knowles 1981) Effects of diflubenzuron

were synergized by profenofos (El Saidy et al 1989) in cotton leafworm fourth instar larvae, and

they were antagonized by 20-hydroxyecdysone (Soltani et al 1987) in beetle (Tenebrio molitor)

pupae More information is needed on interaction effects of diflubenzuron with other chemicals

Table 17.3 Diflubenzuron Effects on Selected Terrestrial Invertebrates

Organism, Dose, and Other Variables Effect Reference a

NEMATODE, Acrobeloides sp.

Fed diet containing 100 mg/kg for 10 days Population reduction of 97% 1

BOLL WEEVIL, Anthonomus grandis

1 µg/female weevil, applied topically After 8 days, about 62% was not absorbed, 3%

was absorbed, and 35% was metabolized and excreted

2

113.4 g/ha, applied 5 times during winter Reduced heavy infestations by >70% in upper

Gulf Coast area of Texas

3

HONEYBEE, Apis mellifera

Fed sucrose syrup/sugar cake diets

containing 0.01, 0.1, 1, or 10 mg/kg for

12 weeks

Adult colony survival reduced at 10 mg/kg;

inhibited reproduction at 1 and 10 mg/kg; no measurable effect on survival or reproduction at 0.01 or 0.1 mg/kg

4

Fed diet containing 10 mg/kg for 10 weeks No reduction in consumption of pollen or in

quantity of brood reared, but >50% reduction in amount of sucrose syrup stored

5

Fed sucrose syrup containing 59 mg/kg for

10 days

Fed sucrose syrup containing 60 mg/L and

drinking water containing 100 mg/L for

40 days

Treated bees consumed significantly less water and pollen and produced significantly less comb, brood, and new workers

7

GERMAN COCKROACH, Blattella germanica

Nymphs fed diets containing 4, 20, 100, or

500 mg/kg for 4 weeks

None dead at 4 mg/kg, 15% at 20 mg/kg, 88%

at 100 mg/kg, and all dead at 500 mg/kg

8

APHID LION, Chrysoperla carnea

0.5 g/L spray Reduced incubation period, reduced hatch, and

reduced survival

9

TERMITE, Coptotermes heimi

Nymphs fed diets containing 100, 500, or

1000 mg/kg

All dead in 24 days at 200 mg/kg, 20 days at

500 mg/kg, or 16 days at 1000 mg/kg Some nymphs developed blister-like swellings on the abdomen and failed to molt into the next instar

10

Table 17.3 (continued) Diflubenzuron Effects on Selected Terrestrial Invertebrates

Organism, Dose, and Other Variables Effect Reference a

MOSQUITO, Culex pipiens quinquefasciatus

Adults fed 500 or 1000 mg/kg diet for 2 days At both doses, 40% of eggs failed to hatch or

hatched abnormally; at the high dose, ovarian histopathology recorded

11

CAT FLEA, Ctenocephalides felis

Larvae, held in rearing medium for

5–6 weeks

TERMITE, Heterotermes indicola

Nymphs fed diets containing 100, 500, or

1000 mg/kg feed

All dead in 14–16 days at 100–1000 mg/kg diet 10

GYPSY MOTH, Lymantria dispar

CABBAGE MOTH, Mamestra brassicae

NEMATODES, various species

Fed diet containing 1 mg/kg for 10 days No effect on reproduction 1

Fed diet containing 10 mg/kg for 10 days 53% population reduction in Panagrellus redvirus,

and 95% reduction in Pelodera sp.

1

AMERICAN COCKROACH, Periplaneta americana

Nymphs fed diets containing 100 or

800 mg/kg for 4 weeks

17% dead at low dose and 52% dead at high dose 8

LARGE WHITE BUTTERFLY, Pieris brassicae

COTTON LEAFWORM, Spodoptera littoralis

Fourth instar larvae, topical application

3, 10, 30, or 100 ng/larva Incorporation of N-acetyl glucosamine into chitin

was inhibited by 23% at 3 ng/larva, 75% at

Adults, 2 species, given 1000 mg/kg diet Fecundity reduced and eggs failed to develop 10

a1, Veech 1978; 2, Bull 1980; 3, Cole 1980; 4, Stoner and Wilson 1982; 5, Nation et al 1986; 6, Muzzarelli

1986; 7, Barker and Waller 1978; 8, Tsuji and Taneike 1988; 9, Zaki and Gesraha 1987; 10, Ahmad et al 1986;

11, Mittal and Kohli 1988; 12, El-Gazzar et al 1988; 13, Martinat et al 1988; 14, Grosscurt et al 1988; 15, El

Saidy et al 1989.

17.4.4 Aquatic Organisms: Laboratory Studies

Studies with diflubenzuron and representative aquatic organisms under controlled conditions(Table 17.4) show several trends:

1 Crustaceans are the most sensitive group of nontarget organisms tested Adverse effects on growth, survival, reproduction, and behavior of copepods, shrimp, daphnids, amphipods, and crabs occur between 0.062 and 2.0 µg/L medium, and early developmental stages were the most vulnerable.

2 Next in sensitivity are aquatic insects, including mayflies, chironomids, caddisflies, and midges Diflubenzuron concentrations between 0.1 and 1.9 µg/L medium produce low emergence and survival.

3 Other groups tested are comparatively resistant (i.e., adverse effects occur at >45 µg/L) In fish, for example, death occurred at >33,000 µg/L.

4 Elevated accumulations occur in aquatic plants during exposure to 100 µg/L and in fish during exposure between 1 and 13 µg/L All species in these groups, however, seemed unaffected by elevated body burdens, as judged by normal growth and metabolism.

The major degradation products of diflubenzuron in water are 4-chlorophenylurea and difluorobenzoic acid (Metcalf et al 1975; Ivie et al 1980) These compounds are less toxic to aquaticorganisms than the parent chemical (Julin and Sanders 1978; Schaefer et al 1979, 1980; Gattavecchia

2,6-et al 1981) A minor m2,6-etabolite, 4-chloroaniline, which is classified as a mutagen by The NationalCancer Institute, and the Cancer Assessment group of the U.S Environmental Protection Agency

(Schaefer et al 1980), is significantly more toxic to fish and Euglena gracilis than is diflubenzuron.

For example, LC50 (96 h) values for 4-chloroaniline and four species of freshwater teleosts are 16 to

56 times lower than comparable data for diflubenzuron (Julin and Sanders 1978), but 4-chloroaniline

is 76 times less toxic than diflubenzuron to Chironomus midge larvae in 48 h (Julin and Sanders 1978) There is a dose-dependent effect of 4-chloroaniline on Euglena growth inhibition and glycine

metabolism in the range of 1 to 200 mg/L during exposure for 30 h (Gattavecchia et al 1981) The

most sensitive organism to 4-chloroaniline was bluegill (Lepomis macrochirus) with an LC50 (96 h)

value of 2.3 mg/L (Julin and Sanders 1978) It is highly unlikely, however, that this concentrationwill be encountered under recommended diflubenzuron application practices

Diflubenzuron inhibits several enzyme systems in crab and insect larvae, resulting in disrupted

glucose metabolism, reduced N-acetylglucosamine incorporation into cuticle, and ultrastructural

deformities of chitinous components of the cuticle (Christiansen and Costlow 1982; Christiansen

et al 1984; Christiansen 1986) Specifically, diflubenzuron inhibits chitin synthetase, a

magne-sium-requiring enzyme that catalyzes the transfer of N-acetyl-D-glucosamine to chitin; the final

result is relatively large accumulations of N-acetylglucosamines (Horst 1981; Machado et al.

at which time many larvae are unable to cast their molts completely and die within a few hours

Several genera of diatoms, including Thalassiosira and Skeletonema, produce up to 33% of their

biomass as chitin These diatoms synthesize chitin strands that extend outside their frustules toincrease buoyancy (Montgomery et al 1990) Chitin-producing diatoms, as well as nonchitanaceous

diatoms, are seemingly unaffected at elevated concentrations of 1 mg/L for periods up to 14 days

(Antia et al 1985) Some species of algae, especially Plectonema boryanum, are reported to

efficiently degrade diflubenzuron (Schooley and Quistad 1979), but this requires verification.Studies with laboratory stream communities dosed for 5 months confirm that insects andcrustaceans are the most severely affected groups Adverse effects occur in the range of 1.0 to1.1 µg diflubenzuron/L Fish and molluscs, however, show no adverse effects at 45 µg/L (Hansen

and Garton 1982) Freshwater clams (Anodonta cygnea) exposed to high concentrations of

difluben-zuron for lengthy periods may experience blocked polycondensation reactions to chitin chains inthe outer mantle epithelium secretory cells, producing unstabilized chitin and increasing shellfragility On this basis, the comparatively resistant burrowing bivalve molluscs may be at risk ifexposed over several calcification periods (Machado et al 1990) Fish accumulated diflubenzuronfrom water up to 160 times water levels, but tissue concentrations during exposure declined steadilyover time (Schaefer et al 1980)

Exposure of Aedes albopictus, a mosquito vector of dengue and encephalitis in Taiwan, for

24 h to 0.00025 to 25 µg/L diflubenzuron resulted in dose-dependent aberrations in larvae, pupae,and adults (Ho et al 1987) (Table 17.4) In general, most treated second and third instar Aedes

larvae died during molting, while most fourth instar larvae developed abnormally (Ho et al 1987).Unfortunately, levels of diflubenzuron used to control saltwater mosquitoes and other insects arealso toxic to zoeal stages of crustaceans (Costlow 1979) and adversely affect growth and repro-duction of adults (Muzzarelli 1986) Treated larvae of estuarine crustaceans are characterized bythe following (Table 17.4) (Cunningham 1986):

• Histological alterations in the cuticular layers of the exoskeleton at concentrations as low as 1 µg/L

• Higher mortality associated with molting and gross morphological deformities at concentrations as low as 0.5 µg/L

• Behavioral modifications at concentrations as low as 0.1 µg/L

Behavioral effects in fiddler crabs (Uca pugilator) were the most sensitive indicator of diflubenzuron

stress, and these effects potentially may influence the ability of juvenile crabs to avoid predation,construct burrows, or feed adequately in nature (Cunningham and Myers 1987) Behavioral effects

on cladocerans that can result in latent mortality include reduced filter feeding rates, reduced bodymovements, and inability to exhibit positive phototaxis, a characteristic of untreated individuals(Cunningham 1986) (Table 17.4) Shrimp larvae exposed to >2.5 µg/L will not undergo dailyvertical migration, and those exposed to 1 µg/L undergo only limited migration, which could affecthorizontal transport and dispersal of populations and reduce recruitment to benthopelagic adultpopulations (Wilson et al 1987) In addition to its inhibitory effect on cuticle synthesis, difluben-zuron affects hormone balance by delaying or arresting the molt cycle, and it inhibits limb regen-eration by inhibiting mitosis and differentiation (Touart and Rao 1987) Regenerated limbs ofdiflubenzuron-stressed crabs that survived ecdysis had lesions in the form of black areas in whichthe cuticle was improperly developed (Weis et al 1987) Also, diflubenzuron caused a reduction

in metabolism of beta-ecdysone in larval insects, leading to an excess of this molting hormone inthe tissues Treatment of decapod crustaceans with ecdysones frequently causes high mortality andmolt acceleration (Gulka et al 1982)

Toxicity and persistence of diflubenzuron in aquatic environments depend on formulation,frequency of application, quantity of organic matter, sediment type, and water pH and temperature.Biological variables are more important than physical variables in assessing diflubenzuron toxicity,especially the age of the test organism and frequency and synchrony of molting during the exposureperiod (Cunningham 1986) Crustaceans and other organisms that molt do not demonstrate a typicalsurvival dose-response curve against diflubenzuron because death occurs only when molting isblocked (Nebeker 1983; Cunningham 1986; Cunningham and Myers 1987; Wilson and Costlow1987) In general, the most sensitive species had comparatively short larval or nymphal periods,

and the organism molted frequently (Rodrigues and Kaushik 1986) Susceptible species include

mayflies (Leptophlebia sp., Baetis pygmaeus), while more-resistant species include a stonefly (Paragnetina media) and caddisfly (Hydropsyche bettani) Amphipods were especially sensitive at

25°C, but not at 10°C, 15°C, or 20°C (Rodrigues and Kaushik 1986)

Mortality patterns of megalops larvae of blue crab (Callinectes sapidus) were elevated at higher

temperatures but were seemingly unaffected by water salinity (Costlow 1979) In studies on larvae

of black fly (Simulium vittatum), diflubenzuron was more effective (1) against earlier larval instar

stages than later ones, (2) against rapidly growing larvae than starved, slow-growing larvae, and(3) at 25°C than at 20°C (Rodrigues and Kaushik 1986) Among diflubenzuron-stressed barnacles

(Balanus eburneus), mortality was higher in fed groups than in starved groups, perhaps due to an

increased uptake from contaminated food or to an increased molting rate due to feeding (Gulka

et al 1980) Increased fragility of cast exuviae from diflubenzuron-treated barnacles suggestsmechanical weakening of the cuticle due to a decrease in chitin content (Gulka et al 1982)

Table 17.4 Diflubenzuron Effects on Selected Aquatic Organisms: Laboratory Studies

Taxonomic Group, Organism, and

Concentration in Medium ( g/L [ppb]) Effect Reference a

ALGAE AND MACROPHYTES

Diatom, Cyclotella cryptica

Blue-green alga, Plectonema boryanum

100 Residues, in µg/kg dry weight, during exposure for

4 days were 144,700 at 1 h, 85,700 at 1 day, 56,900

at 2 days, 11,700 at 3 days, and 8300 at 4 days;

Plectonema growth rate was unaffected

2

Alga, Selenastrum capricornutum

Diatoms, 3 species (Skeletonema

costatum, Thalassiosira, nordenskioldi,

Hydra, Hydra oligactis

0.1–0.12 (estimated) After 24-h exposure, asexual budding rate significantly

increased over controls during 20-day posttreatment period; some histopathology Second-generation hydras not significantly different from controls

4

PLATYHELMINTHES

Planarian, Dugesia dorotocephala

5 No effect on survival, behavior, or asexual reproductive

capacity after 24-h exposure

23

AQUATIC INSECTS

Mosquito, Aedes aegypti, 4th instar larvae

20 (equivalent to 0.056 kg/ha) Fatal to 100% within 24 h, about 50% after 4 days,

and <20% after 8 days

0.125 Histopathology of cuticle and anal gills in 4th instar

larvae after 24-h exposure of 3rd instar larvae

6 0.21 Adult emergence inhibited when 3rd instar larvae

Caddis fly, Clistoronia magnifica

Midge, Cricotopus spp.

4.9 Molting and survival adversely affected during

exposure for 96 h

11

Mosquito, Culex pipiens, exposed as

4th instar larvae for 24 h

Mosquito, Culex pipens quinquefasciatus

1.0 Fourth instar larval dip had no effect on adult sterility 13

Chironomid, Glyptotendipes paripes,

Blackfly, Simulium vittatum, larvae

80 for 30 min at various water

temperatures

Table 17.4 (continued) Diflubenzuron Effects on Selected Aquatic Organisms: Laboratory Studies Taxonomic Group, Organism, and

Concentration in Medium ( g/L [ppb]) Effect Reference a

Stonefly, Skwala sp.

Midge, Tanytarsus dissimilis

4.9 Molting and survival adversely affected during 5-day

exposure

11

ARACHNOIDS

Horseshoe crab, Limulus polyphemus

5 Larvae exposed for 24 days showed slight delay in

molting at 14 days; survival as in controls

6

50 Larvae exposed for 24 days showed molt rate as in

controls, but high mortality immediately after ecdysis;

reduced growth of survivors

16

MOLLUSCS

Clam, Anodonta cygnea

200,000 After exposure for 3 months, all clams survived and

appeared healthy But normal calcification process disrupted on lamellar layer of the shell, producing fragile shell

17

Snail, Juga plicifera

36–45 No effect on survival, growth, or reproduction during

3-week exposure

3, 11

Snail, Physa spp.

45 No measurable effect on growth, survival, or

reproduction during 3-week exposure

3, 11

CRUSTACEANS

Copepod, Acartia tonsa

Adults exposed following terminal molt

1 Hatch of viable nauplii reduced by 50% after 12-h

exposure; no hatch after 36-h exposure Effect not reversible for at least 30 h after exposure

18

10 Hatch of nauplii reduced by >95% after exposure for

24 h and 100% after 36 h Effect not reversible for at least 26 h after exposure

18

100 No effect on egg production during 14-day exposure 18

1000 No adverse effect on survival during exposure for

5 days

18

Brine shrimp, Artemia salina

Adults exposed to 1, 2, 5, or 10 During exposure for 80 days, there was a significant

reduction in reproductive lifespan at 2, 5, and 10 µg/L

Nauplii produced viviparously by mated pairs were comparable to controls — except for the 10-µg/L group, which produced fewer nauplii Cysts produced oviparously by treated pairs, however, had lower mean hatchability

19

Nauplii exposed to 1, 10, or 100 All dead within 30 days in the 100 µg/L group; survival

same as controls in 12 days for the 1 and 10 µg/L groups

19

Barnacle, Balanus eburneus

50–100 Significant acceleration of intermolt cycle at low dose,

and among survivors at high dose

21

750 or 1000 High mortality during 10-day exposure; prolonged

premolt; histopathology of cuticle-secreting epidermal cells

1000 for 48 h plus clean seawater for

Blue crab, Callinectes sapidus

Ostracod, Cypicerus sp., Cypridopsis sp.

Daphnid, Daphnia magna

0.062 Survival and reproduction adversely affected in full life

cycle (21-day exposure)

1000 After 30-min exposure, 91% dead in 9 days at 25°C 14

Amphipod, Hyalella azteca

2–1000 Prolongation of copepodite stage for 3–4 days,

followed by death without molting, in most cases At

125 µg/L and higher, partial molting occurred but all died

25

1000 LC50 (48 h) for copepodites No effect on mating

behavior of adults but abnormal ovisac development and decreased fecundity in some females

25

Mysid shrimp, Mysidopis bahia

0.075 Reduction in number of young per female after

exposure for 21 days

26 0.075 Reduced survival and reproductive success after

exposure for 28 days

1.9 Exposure for 24 h resulted in 65% mortality 3 days

after treatment; progeny produced before death had

a significantly lower reproduction rate than controls,

as did those in the next generation at nanogram/L (ppt) concentrations

27

Grass shrimp, Palamonetes pugio

0.1–0.5 No effect on duration of molt cycle, but dose-related

inhibition of regenerative limb growth noted (EC50 = 0.11 µg/L for left 5th periopod)

during or immediately after molting

29

<1.0 Almost all larvae that survived to day 15 eventually

metamorphosed successfully to postlarvae

32 1.0 (initial), medium aged for 71 days,

with or without sediments

No deaths of larvae in 22 days when sediments present; all larvae died within 22 days when sediments absent

34

1.4 LC50 (96 h) larvae, 95% confidence interval (CI)

1.27–1.54, for wettable powder (WP-25) in water

negative phototaxis suppressed

29 3.4 LC50 (24-h exposure, held until molting complete in

24–48 h)

29 1.0 (initial) Fatal to larvae exposed to medium aged for 71 days

without sediments; no deaths of larvae after exposure for 22 days to medium aged for 71 days with sediments (about 0.5 µg/L)

34

202 LC50 (96 h), adult males and nonovigerous females 33

2000 Negligible mortality of late premolt stage during

exposure for 96 h

29

Crab, Rithropanopeus harrisii

0.05 No effect on positive phototaxis response of stage IV

larvae

35

0.3–0.5 Increased swimming speed of stage I, II, and III larvae 35 0.5 No adverse effects on larval survival during exposure

for 20 days

36

10 All larvae died during 32-day exposure in containers

without sediments; containers with sediments were

no longer toxic after 19 days

34

Crab, Sesarma reticulatum

1 No adverse effects on larval survival during 40-day

exposure

36

Table 17.4 (continued) Diflubenzuron Effects on Selected Aquatic Organisms: Laboratory Studies Taxonomic Group, Organism, and

Concentration in Medium ( g/L [ppb]) Effect Reference a

Copepod, Tigriopus californicus

0.1–100 During 72-day exposure, no adverse effects were noted

on adult survival and juvenile development at 0.1 µg/L

Reproduction was inhibited at 1 and 5 µg/L Copepods exposed to 10 or 100 µg/L did not reproduce, were moribund, and had decreased survival.

1

Tadpole shrimp, Triops longicaudatus

Fiddler crab, Uca pugilator

Juveniles exposed for 24 h once a week

for 10 weeks, then held in clean

seawater for an additional 14 weeks

0.2 No adverse effects on survival or ability to escape from

20 All died in 23 weeks; reduced mobility prior to death 37

200 All dead in 8 weeks; most deaths occurred in first

4 weeks

37 Adults exposed to 0.5, 5, or 50 after

multiple autotomy of one chela and

5 walking legs

Continuous exposure for 18 days produced a dependent retardation of regeneration and deaths during molt at 5 and 50 µg/L The presence of sediment in test containers lessened effects, but did not eliminate them

dose-38

Adults exposed for 1–3 weeks

0.5–50 Some reduction in number of burrows dug at 15 and

60 min after exposure

39 Unknown Burrowing activity normal on sediments containing

Mosquitofish, Gambusia affinis

Unknown Fish exposed to radiolabeled diflubenzuron for 33 days

contained about 6% of the parent diflubenzuron vs

54% for alga (Oedogonium cardiacum), 82% for snail (Physa sp.), and 94% for larvae of mosquito (Culex

43

Brown bullhead, Ictalurus nebulosus

13.2 in pond surface layer 1 h after

treatment, <0.2 after 14 days

Maximum concentrations, in µg/kg whole-body fresh weight (FW), were 387 at day 1, 190 at day 2, 42 at day 4, and ND at day 7

44

Channel catfish, Ictalurus punctatus

Unknown Runoff from soil containing 0.55 mg diflubenzuron/kg

at start produced maximum residues during 28 days,

in µg/kg FW, of 4 in muscle and 10 in viscera

2

Bluegill, Lepomis macrochirus

1–10 Bioconcentration factor of 13–20 after 24-h exposure 45 2.5 Growth reduction of 56–86% for young of year held in

large enclosures for 3 months — attributed to reduction in food items