Laboratory and field experiments with methoxychlor as a larvicide for simuliidae (diptera)

LABORATORY A N D FIELD EXPERIMENTS WITH METHOXYCHLOR AS A LARVICIDE FOR SIMULIIDAE (DIPTERA) RON R. WALLACE

Environment Canada, Freshwater Institute, 501 University Crescent, Winnipeg, Manitoba, Canada, R3T 2N6 H. B. N. HYNES

Dept. of Biology, University of Waterloo, Waterloo, Ontario N2L 3Gl, Canada &

W F. MERRITT

Atomic Energy of Canada Ltd, CRNL, Chalk River, Ontario, Canada ABSTRACT The dispersion of methoxyehlor and Rhodamine B dye in the Chalk River, Canada, was measured by chromatography and fluorometry. The accumulation of methoxychlor was studied in laboratory and field experiments. The river was treated for 15 rain with an oil solution of metho xychlor (0.79 I~g/litre ). The numbers of drifting simuliid larvae reachedmaximum values 60 min after the start oj" treatment at stations 275 m and 550 m downstream from the point of application. The peak numbers decreased with distance downstream, and most of the larvae began to drift after the methoxychlor had passed by. No methoxychlor could be detected in water, larvae or moss collected from the Chalk River before the experiment, but drifting larvae caught after the treatment contained residues ranging from 0.24 to 2.57 mg/kg. The larvae which began drifting later generally containedmore methoxychlor than those drifting soon after treatment. S. v e n u s t u m began drifting sooner than S. vittatum, and the mean sizes of drifting larvae of both species tended to increase as time passed. Residues of methoxychlor ranging from 7.4 to 34-6 Itg/kg were detected in moss and grasses in the river for up to 8 weeks after the treatment. Laboratory experiments indicated that simuliid larvae concentrate particulate formulations of methoxychlor more efficiently than ethanol ones. Residue values in the larvae reached 2310 I~g/kg. Trichoptera larvae concentrated methoxychlor to levels up to 1563 I~g/kg, and ethanol formulations to higher levels than particulate ones. 251

Environ. Pollut. (10)(1976)--© Applied Science Publishers Ltd, England, 1976 Printed in Great Britain

252

RON R. WALLACE,H. B. N. HYNES, W. F. MERRITT INTRODUCTION

Chance (1970), Jamnback (1973), M uirhead-Thompson (1971), and Wallace ( 1971, 1973) have reviewed assessments of the impact of pesticides on so-called 'target' and 'non-target' species of aquatic animals. However, apart from the problems associated with estimations of biomass, comparisons of the impact of pesticides in running waters are complicated by the variability in types of stream in which the Simuliidae breed. In addition, Simuliidae may comprise an important segment of the available food of trout (Salvelinusfontinalis (Mitchell)) (Power, 1966) and possibly of some invertebrates (Peterson & Davies, 1960). Although the degradation and transport ofmethoxychlor ( 1, 1, 1-trichloro-2,2-bis (p-methoxyphenyl)ethane) in water has been extensively studied (Burdick et al., 1968; Kapoor et al., 1970; Kennedy et al., 1970; Metcalf et al., 1971; Merna et al., 1972), this has been done mostly in lentic situations. Although methoxychlor has been noted for its impact on simuliids (Jamnback, 1973) less information on its degradation is at present available for lotic ecosystems (Helson, 1972; Kruzynski & Leduc, 1972; Wallace et al., 1973a, b; Fredeen, 1974). This study was therefore designed to investigate the behaviour of methoxychlor in a river and its persistence and accumulation in biota found in running waters.

MATERIALS AND METHODS Field experiments Field experiments were carried out in the Chalk River, Ontario, Canada (40 o 01' N, 77 ° 24' W) where sampling stations were 275, 550 and 770 m downstream from the treatment point, which itself was 50 m below the outflow of a large lake and was the start of a series of rapids in the river. The river flowed rapidly throughout the study reach, except for a large pool (11 m × 24 m), immediately downstream from station 550 m. Total discharge was estimated by dye dilution to be 2820 litres/sec. Some water chemistry analyses were made in the field with a Hach Kit, but a Beckman pH meter was used to determine pH and nitrate was determined in the laboratory (Table 1). Methoxychlor was diluted in fuel oil from a stock oil solution containing 150.2 g technical methoxychlor plus ½~o by weight of emulsifier (Triton X-161) per litre. The methoxychlor, together with an aqueous solution of Rhodamine B dye, was metered into the stream at a constant rate for 15 min using methods similar to those described elsewhere (Wallace et al., 1973a). The calculated initial concentrations of methoxychlor and Rhodamine B in the river were 0.79/~g/litre and 1-0/~g/litre, respectively. Pairs of drift nets made of Nitex nylon netting (253-67 x 23 threads/cm and 30 × 30 cm and 75 cm long) were bolted side by side and attached in mid-channel at each station by clamps fastened to a rope strung across the stream. The nets at all

EXPERIMENTS WITH METHOXYCHLORAND SIMULIUM

253

stations were replaced and emptied at 15-min intervals beginning 15 min before the start of the experiment. Their contents were washed into large pans, strained, and placed into plastic bags, and shortly afterwards frozen to - 20 °C, being kept at this temperature until analyses were carried out. Water samples were collected at all stations at intervals throughout the experiment. At station 275 m concentrations ofRhodamine B were read directly with the fluorometer. Samples from the other station were evaluated with the fluorometry techniques described elsewhere (Wallace et al., 1973a). Earlier experiments had shown that neither the fuel oil nor methoxychlor interfered with the determinations by fluorometry. The water samples collected for analysis of insecticide were frozen in polyethylene bottles and shipped to Dr J. R. Duffy (University of Prince Edward Island) for gas chromatographic determinations of methoxychlor. TABLE 1 PHYSICAL--CHEMICAL DATA FROM THE CHALK RIVER

4 J U N E 1972

Temperature: 19°C pH: 7-3 Total alkalinity (ppm calcium carbonate): 12mg/litre Calcium hardness (EDTA method): 12.5mg/litre Total hardness (EDTA method): 18.5mg/litre Magnesium hardness: 6.0mg/litre Nitrate (Phenoldisulphoricacid method): 0.18mg/litre Dissolved oxygen: 8.4 mg/litre (73 ~) saturation Phosphate (ortho): 0.05mg/litre Silica: 11.3mg/litre Turbidity: 21 JTU Suspended material ranged from 1.5-3.6mg/litre Air temperatures and precipitation: (see Fig. 6) Immediately before the treatment, samples of moss (Hygrohypnum luridum (Hedw.) Jenn.), Fontinalis novae-angliae (Sull.), Fissidens osumundioides (Hedw.)), and grass (Cypruss sp.) were collected from points near the middle and along the sides of the Chalk River 275 and 550 m downstream from the point of treatment. They were stored a t - 20 °C. The invertebrates from one drift net (at each station) were used for the gas-liquid chromatography (GLC) analyses and numerical determinations, and those from the other for species identifications and size measurements. The samples were thawed and simuliid larvae picked from the detritus under a binocular microscope. However, in order to limit decomposition of the methoxychlor, the larvae were kept on ice. The average time for picking ranged from six to eight hours per sample, after which time the larvae were again held a t - 20 °C. Larvae used for size measurements and specific identifications were sub-sampled according to the method of Strickland (1954). Measurements were made with a micrometer. Extractions of methoxychlor from the samples were begun 3 weeks after the treatment. The larvae were thawed and centrifuged at a low speed in a Nitex bag to

254

RON R. WALLACE,H. B. N. HYNES,W. F. MERRITT

remove water (Howmiller, 1972). They were then weighed and homogenised with 1 : 2 w/w anhydrous sodium sulphate to remove remaining water. Glassware and solvents used for the GLC analyses met chromatographic grade standards of cleanliness (MacLeod et al., 1969). Following a 6-h Soxhlet extraction in n-hexane, the extract was eluted and evaporated just to dryness in a rotary evaporator. Cleanup procedures were similar to those used by Langois et al. (1964). Samples of vegetation were thawed, washed with distilled water to remove sand and debris, centrifuged at low speed to remove water, and weighed. They were then ground for 5 min at high speed in a metal Sorval homogeniser in a solution of 2:1 acetonitrile :water. The extract was filtered through a medium porosity sintered glass funnel into a separating funnel. Then 100 ml n-hexane were added and the sample was shaken for 1 min and allowed to stand. When phase-separation had taken place, 10 ml of a saturated salt water solution and 300 ml of water were added. The aqueous layer was discarded and the hexane extract passed through a 2-3 cm deep layer of anhydrous sodium sulphate. Rinsings from the separating funnel and sodium sulphate were added to the extract and evaporated just to dryness in a rotary evaporator at 45 °C. Residues were redissolved in 5 ml n-hexane and stored at --20 °C. Column cleanup procedures for the vegetation were identical to those used for the animal tissue extracts. The gas chromatograph used was a Perkin-Elmer Model 900 equipped with a Ni 63 electron capture detector. Column and operating conditions were: Column: 6' × 6mm OD glass with 6 ~ QF-1 Chrom. W A/W 80/100 Column temperature: 195 °C Injector temperature: 245 °C Nitrogen flow: 120 ml/min Before use the column was conditioned for 2 h at 245 °C. Calibrations and peak identifications were made using Nanogen pesticide analytical standards of methoxychlor (10ppm w/v + 0.5 ~) obtained from BDH Chemicals Ltd, Poole, Great Britain. Injections were made with a Hamilton microlitre syringe. Analytical standards of methoxychlor were interspersed between the unknown samples, and in many samples both were mixed and chromatographed together once as a further aid in peak identification and quantification. Also, each unknown sample was chromatographed alone at least three times and was quantified as an average value of the resultant peak heights. Uncontaminated stream moss and simuliids, extracted and chromatographed together with known amounts of methoxychior analytical standard, indicated that extraction efficiencies ranged from 76 to 84 ~. In another experiment, which did not involve any insecticide, an attempt was made to simulate an aerial spraying by monitoring concentrations of Rhodamine B

EXPERIMENTS WITH METHOXYCHLORAND SIMULIUM

255

applied to the river. Men were positioned in mid-stream 550 m, 275 m and just upstream from the fluorometer, and the experiment was co-ordinated by means of portable two-way radios. Spraying of the dye solution into the river was begun 2, 1 and 0 rain after a given signal at the stations 550 m, 275 m, and just upstream of the fluorometer, respectively. Hand pumped atomisers were used to apply 30 ml of a 5 ~o Rhodamine B dye solution as a spray mist over a period of 90 sec. The spray was applied as widely across the stream as possible, so that the whole experiment was an attempt to simulate an aerial spray of the stream with swaths centred on 275 m crossings occurring progressively upstream at intervals of I min.

Laboratory experiments Larvae of Simulium vittatum (Zett.) and Hydropsychidae (mostly Cheumatopsyche but some Hydropsyche) were collected from the Cananagigue Creek near Floradale, Ontario, Canada, and placed in separate circular glass aquaria containing 1000 ml of deionised water. The water was aerated, maintained at room temperature and stirred at low speed with a magnetic stirrer. The larvae were left to acclimate for 24h before the exposure period. By then, the Simulium had become established along the lines of flow on the sides, and the Trichoptera had spun nets among small pebbles. Most of the Simulium were in the last larval instar; the Trichoptera were in the penultimate larval instar. Twelve aquaria were used (six each for the Trichoptera and Simuliidae). Separate tanks were used as control, 15 and 30 min exposures, respectively, for each of the two pesticide formulations used, namely ethanol solution or particulate. After the 15 and 30 rain exposures larvae were removed, rinsed twice in distilled water, and then centrifuged at low speed to remove water, weighed and frozen t o - 2 0 °C. Calculated methoxychlor concentrations of 0.075 and 0.1 mg/litre were administered to exposure tanks for the particulate and ethanol formulations, respectively. The ethanol solution of methoxychlor was made up from recrystallised technical methoxychlor. Confirmation of purity was obtained by melting point determinations and gas chromatography. Particulate methoxychlor was obtained from Johns-Manville Research & Engineering Center, Manville, New Jersey, USA via Dr A. S. West. The micropulverised compound consisted of 63 ~ active methoxychlor made up as: 70 ~o Technical methoxychlor oil concentrate (90 ~ ) 25 ~o Micro-cel E 1-5 ~o Igepon T-77 3.5 ~o Polyfon T. An industrial 'Model B' coulter counter was used to obtain measurements of particle size spectra of the particulate methoxychlor. The insecticide was briefly agitated in Isoton diluent and replicated measurements were made with the 200/~ aperture.

256

RON R. WALLACE, H. B. N. HYNES, W. F. MERRITT

Measurements of the size spectrum of the particulate methoxychlor indicated that particle diameters ranged in size from 4-2 to 33.7 #, but that the (numerical) majority of particles ranged from 5 to 20 #. The lower measurement (4.2/~) is probably conservative as measurements of smaller particles were thwarted by clogging of the smallest aperture of the counter by the larger particles also present.

2.5

2.0

%

t'Y

0

10 2O 30 40 5-0 TIME (MINUTES) FROM BEGINNING OF TREATMENT

Fig. 1. Concentrations of Rhodamine B dye in the Chalk River after the simulated aerial spray.

RESULTS Air spray simulation

The results of the simulated air spray (Fig. 1) indicate three successive peaks of Rhodamine B dye with the first, from the sprayer closest to the fluorometer, rapidly reaching and falling from the maximum values recorded; decreasing peak concentrations occurred with successive maxima. Also, the increasing times of passage for peaks resulting from additions made progressively further upstream were similar to those reported in previous experiments (Wallace et al., 1973a). Dye was detected in the stream water for almost 50 min after the sprayings.

257

EXPERIMENTS WITH METHOXYCHLOR A N D SIMULIUM

Stream treatments

Dispersion of dye, methoxychlor and drifting simuliids: At station 275m Rhodamine B dye, although apparently proceeding and remaining in the water column somewhat longer than methoxychlor, reached maximum values at almost the same time as the insecticide (Fig. 2). This did not happen at the two downstream stations. At station 550 m although the dye was clearly detected, no methoxychlor was found in the water at any time. At station 770 m peak values for both dye and methoxychlor were detected but at different times. Concentrations of dye in the stream decreased continuously while increasing in times of passage with distance downstream. Simuliid larvae comprised most of the invertebrate drift, together with

300

"\

L.

\

"4

Station - 275 m e t r e s 200

c

\ I[7

Q

100 i

\, \.

v

m {-

" m " ~ •- ~ 3 ~ . ~ . _ . ~ _ . _ , . ~ _ . _

-[: "I[3 0

# 0,8 x~ Q6 r

S t a t i o n - 5 5 0 metres

.200e~E

/ .-O.k //

'\'\

>

0.4

"100

m o.2

>~

ru x 0r -

Station - 7 7 0

N 0.6

metres

'12°°

"5

I

:k

..... I

02

310 6'0 Time (minutes)

9'0 12'0 15'0 180 f r o m beginning of treatment -

Fig. 2. Concentrations of methoxychlor and Rhodamine B dye and numbers of drifting blackfly larvae in the Chalk River after the treatment at the three sampling stations.

258

RON R. WALLACE, H. B. N. HYNES, W. F. MERRITT

only small amounts of Trichoptera (Cheumatopsyche and Hydropsyche) and Ephemeroptera (Ephemerella) and some Sialis. The numbers of drifting simuliid larvae reached peak values 60 min after the beginning of the treatment at stations 275 m and 550rn, and somewhat later at station 770 m (Fig. 2). As with the dye, the peaks for simuliid larvae decreased with distance downstream while taking longer to pass by. There was also a clear separation of peak values for Rhodamine B and the drifting larvae, the latter reaching peaks later than the dye at all stations.

3001

9//~

Station - 275 metres

::t / /

I3'0

It

oo .O o I! 15o

/

\

• /

©

m

--2501

Station -550 metres

[

2004

~

2~

~1~ol

~

II

J o

t

IHIII

~°1 Z

>

/I I I I--ILI I 0

6

9

10

/ 10

10

Time (minutes) from beginning ot treatment Fig. 3. Numbersof drifting blackfly larvae and the concentration of methoxychlor in the larvae at various times after the treatment.

Methoxychlor residues in simuliid larvae: No methoxychlor was detected in larvae gathered at station 275m before the experiment, but larvae caught after the treatment in the nets 275m and 550m downstream had concentrations of methoxychlor that generally increased with time (Fig. 3). No analyses were made on larvae from station 770 m because so few drifted by there. Residues in larvae caught

EXPERIMENTS WITH METHOXYCHLOR AND SIMULIUM

259

at station 275 m varied more erratically than those from station 550 m, and during the maximum drift were generally lower at station 275 m (Fig. 3). Species and size composition o f the drifting simuliids: Samples of larvae collected from rocks and in drift nets both before and after the treatment showed that S. venustum (Say) and S. vittatum (Zett.) were by far the most common species. Samples taken from nets after the treatment indicated that at both station 275 m and at station 550 m the numerical proportion of drifting S. venustum generally declined with time, while that ofS. vittatum increased (Fig. 4). Also, the mean sizes of both species increased with time at both stations (Fig. 5). An analysis of variance (FTest) indicated that successive mean size values were significantly different (P < 0.01) at each station, and that there was a significant tendency for mean values

1:: t -

~.o r~.~_ ~ 50 a.

L

o

o

_i t J 60

120

180

50

:o o

T I M E (minutes)

Fig. 4. Percentagecompositionof the drifting Simuliidae(by species),'darkbars indicate S. vittatum and light bars S. venustum in the Chalk River after the treatment. to rise with time. In addition, the mean sizes of S. venustum were generally larger than those of S. vittatum at similar times, with values for each species tending to be higher at the upstream (275 m) station than at the downstream one. No simuliid larvae could be found on rocks and moss in the treatment zone 24 h afterwards. It may also be noted here that substantial quantities ofRhodamine B dye occurred in the gut and on the anterior end of the cuticle of many large simuliids. The presence of the bright scarlet dye in the gut suggests that the larvae had eaten it. Methoxychlor residues in vegetation." While no methoxychlor was found in moss and grasses collected before the experiment, residues occurred in samples from stations 275 m and 550 m up to 8 weeks after the treatment (Fig. 6). The amounts at

260

RON R. WALLACE,H. B. N. HYNES, W. F. MERRITT

station 275 m tended to decline as time progressed, but there was no very clear trend at either station.

Laboratory experiments Simuliidae." Simuliid larvae exposed to methoxychlor for 15 and 30 min in the laboratory showed considerable accumulations of insecticide. Larvae from control

275m

S. venus't~m

4

hi

t

3

t

t

t

t

> rr <( ..J t3

1275 m S_.vittaturn

I

_J

t

i

(.9 Z

t

[

24

I

h n~ (3 LL

0

g E

550 m S. venustum

it 24

tZ) hi

m S. vittatum

L/)

t

Z

hi 0

t

6b ~o TIME (minutes)

~ao

Fig. 5. The mean sizes of drifting Simuliidae (mm) at various times after the treatment with methoxychlor. Points indicate the mean, the bars indicate the standard deviation. tanks had no detectable residues, but those held in particulate or ethanol solutions showed values ranging from 30.8 (2310 #g/kg) to 20.7 (1566 #g/kg) and from 0.82 (82#g/kg) to 6.9 (688 #g/kg) times the exposure levels of the two treatments, respectively (Fig. 7). The concentration from the particulate preparation was much greater than that from the ethanol solution (Fig. 7), but it appeared to increase with time of exposure for the latter and decrease for the former.

261

EXPERIMENTS WITH METHOXYCHLOR AND SIMULIUM

AIR

ou

TEMPERATURE

15

1

.5

PRECIPITATION

u

50 S T A T I O N - 275

METRES

,7, 40 3O z

o

7

'--

2O 10

>

o

_1 I t,lu S T A T I O N - 550

METRES

u 50 z

o

40

3C 2O 10

I 2-

,11 I , I I , 7

14

JUNE

21

28 Jl

7

14

JULY

21

28

ll

7

14

AUGUST

Fig. 6. Air temperatures and precipitation near the Chalk River I June-14 August 1972. Concentration of methoxychlor residues (#g/kg) in vegetation (wet weights) taken from the Chalk River at various times after the treatment. No residues were detected in samples taken before the treatment. The circled T indicates the date of the treatment.

262

RON R. WALLACE, H. B. N. HYNES, W. F. MERRITT

Trichoptera; Although levels of residues from the particulate formulation were higher than those from the ethanol solution in Simuliidae, the reverse was true for Trichoptera (Fig. 7). As with Simuliidae, larvae from control tanks had no detectable residues; however, values in treated larvae ranged from 15.6 (1563 #g/kg) to 14.5

TRICHOPTERA

SIMULIIDAE O'J

L I0

.E U

1®

v

15 L

o

I

15 EXPOSURE TIME(mIn)

I

30

30

I

J~ u X 0 .C

30

~

2o

t-

.g 0

u

10

E

I[

0

15

I

30

EXPOSURE

15

I

30

I

TIME (min)

Fig. 7. Concentration of methoxychlor in Simuliidae and Trichoptera larvae after the laboratory exposures. Dark bars indicate the animals exposed to the particulate formulation and light bars those exposed to the ethanol formulation. Ordinates: (upper) the concentration of methoxychlor (#g/kg) wet weight basis, in the larvae; (lower) the magnification of methoxychlor in the larvae above the maximum calculated concentrations in the water.

(1453 #g/kg) and from 8.2 (615 #g/kg) to 10.4 (782 ~g/kg) times above exposure levels for the ethanol and particulate formulations, respectively. Residues from the ethanol solution were slightly lower after 30 min than after 15 min, but they appeared to increase slightly with time in the exposure to the particulate formulation.

EXPERIMENTS WITH METHOXYCHLORAND SIMULIUM

263

DISCUSSION Simulation of aerial sprays The profiles of concentration of Rhodamine B in the Chalk River which resulted from the treatments with the hand sprayers were probably similar to those which occur in streams after commercial aerial sprayings. However, it should be noted that the swaths produced by the hand sprayers were probably narrower than those from aerial sprays, and there are certainly differences in droplet sizes and times of descent to the stream. In any case, the general trend of decreasing concentration maxima and increasing times of passage of peaks with distance downstream, as observed in the simulation, would almost certainly be produced by commercial aerial larviciding which cross streams at 400m intervals. If so, stream invertebrates would be exposed, for a short time, to a sudden maximum concentration, and then to additional lower concentrations which would take longer to pass downstream. The ground treatment with methoxychlor and Rhodamine B dye Earlier speculation that oil solutions of insecticides when poured into streams from the ground do not become uniformly distributed in the water (Jamnback & Frempong-Boadu, 1966) may be confirmed by this study. Maximum concentrations of Rhodamine B and methoxychlor occurred simultaneously at station 275 m, but the detection of dye both before and after the methoxychlor suggests incomplete mixing of the insecticide in the stream. No methoxychlor could be detected in water samples from station 550m although Rhodamine B was clearly present, so it is possible that the soluble dye and the less soluble oil formulation of insecticide had become separated in the stream. However, it seems unlikely that the separation was complete in view of the uniformly rapid flow between sampling stations 275 m and 550 m and previous work carried out in similar streams over even greater distances (Wallace et al., 1973b). Water samples from station 550 m used for fluorometric and chromatographic analyses were taken from the inside of a sharp bend in the river. It is possible that the oil formulation of insecticide followed the faster flow of water towards the outside of the bend and that the more soluble dye was more widely dispersed in the channel, thus being included in the water samples. In any event, the dye, and possibly methoxychlor as well, continuously decreased in peak concentration and peaks took longer to pass by with distance downstream. It was also clear that the two chemicals had become separated at station 770 m, the pesticide being detected in'patches' well after the dye had passed (Fig. 2). This separation was probably caused by the large pool downstream from station 550m which probably reduced the effectiveness of the pesticide. The separation of the dye and the oily pesticide by the large pool could be expected in view of the differences in solubilities of the two chemicals, but the absence of methoxychlor in samples from the faster waters at station 550 m was not anticipated.

264

RON R. WALLACE,H. B. N. HYNES, W. F. MERRITT

This brings into question the application of earlier preliminary studies (Wallace et al., 1973b) of the use ofRhodamine B for monitoring insecticides in running waters, at least where extremely low dosages are involved. It seems clear that oil formulations of insecticides do not disperse uniformly in streams, a finding which may explain the occurrence of blackfly larvae after similar treatments in other streams (Wallace, 1971). The drift of invertebrates after treatment The numbers of simuliid larvae drifting past each sampling station rapidly increased to, and then declined from, maximum values (Fig. 2). A similar pattern of drift 300 m downstream from an aerial spray with DDT was reported by Collins et al. (1952). Methoxychlor causes a steady and quick release of blackfly larvae from the substrate; Jamnback & Frempong-Boadu (1966) found that it detached more than 90 ~o within 30 min of an exposure of 0.4 mg/litre for 5 min. The pattern of the catastrophic drift of simuliid larvae after the treatment closely resembled that of the dye, with maximum values decreasing downstream and peaks taking longer to pass. As no larvae could be found in treated areas 24 h after the experiment, the drift of most of the entire population was probably recorded. The basic mixing processes of flow are important in the dispersal of various materials in streams, but the decreasing numbers of drifting larvae with increasing distance downstream may also have been caused by reattachment of moribund larvae and consumption by fish. Such effects may have been produced by large pools below station 550m and may perhaps account for the very low catch at 770m (McLay, 1970). Muirhead-Thompson (1971) speculated that an insecticide which induced rapid detachment of larvae would be particularly effective because of the longer time of exposure endured by the insects floating downstream with the insecticide. However, our results indicate that most of the drift occurred well after the passage of the insecticide at station 275 m and probably at station 550 m as well. Hence, it seems doubtful that the mechanism proposed by Muirhead-Thompson is important in poisoning larvae, even with insecticides which cause larvae to detach quickly. Possible differential susceptibility of larvae to methoxychlor The fact that larger numbers of S. venustum began to drift more quickly than S. vittatum (Fig. 4) may indicate different susceptibilities of the two species to methoxychlor. Also, as the mean sizes of the larvae of both species increased progressively with time after the treatment (Fig. 5), the smaller individuals may be more affected than the large ones. The reasons for such varying susceptibilities may include factors such as different rates of feeding, and different physiological tolerances of various species and instars. Such differences in susceptibilities to pesticides among aquatic invertebrates have been reported previously. Gjullin et al. (1950) suggested that older larvae of Simuliidae are more tolerant of DDT.

EXPERIMENTS WITH METHOXYCHLORAND SIMUL1UM

265

Jamnback & Frempong-Boadu (1966) found that after 5min laboratory exposures to 0.4 mg/litre methoxychlor slightly fewer mature larvae had detached than had smaller sized specimens. Jensen & Gaufin (1964), working with two species of Plecoptera and several insecticides, found that the resistance of one to DDT was greater than that of the other, and that larger specimens were more tolerant to several poisons. The accumulation of methoxychlor by Simuliidae and Trichoptera There is growing evidence that ingestion may be important in the uptake of insecticides by simuliid larvae. After the enhanced kill of larvae caused by the adsorption of DDT onto suspended clay and silt (Fredeen et al., 1953a, b) other workers mixed DDT with clay and hard silt (Noel-Buxton, 1956) and achieved similar results, especially when extra particulate matter was present in the streams. Travis & Wilton (1965) also discussed the possible effects of particle feeding by simuliid larvae on the efficacy of pesticides. Studies in Britain using water insoluble particles of DDT (Kershaw et al., 1965, 1968) and more recently in Canada with particulate methoxychlor and abate (Helson, 1972) attest to the effectiveness of these formulations. It appears that methoxychlor, like DDT, is also quickly adsorbed onto particles in water (Merna e t al., 1972). The efficiency of feeding may have contributed to the different concentrations of methoxychlor found in larvae exposed to the pesticide in the laboratory and in the field. In our laboratory experiments larvae concentrated the ethanol formulation of methoxychlor to levels ranging from 82 to 688 #g/kg, and much higher values were found with the particulate formulation (1556-2310/~g/kg). Residues found in larvae after the treatment of the Chalk River ranged from 240 to 2570 #g/kg, even though the maximum concentration of methoxychlor in the water was much lower (0.79 #g/litre) than in the laboratory experiments with the ethanol (100 #g/litre) or particulate (75/~g/litre) formulations. This suggests lhat accumulation may be even more efficient with the oil solution used in the field than for the particulate formulation used in the laboratory. This may be because the positioning of larvae for feeding was better in the natural situation than in the laboratory. Also, perhaps, the turbulent flow in the river may have presented more particles in preferred size ranges than in the laboratory. The concentration of methoxychlor used in our field experiment (0-79 pg/litre) was much lower than that used (0.075mg/litre) in commercial ground-level operations, and is the lowest effective dosage for a ground-level larvicidal operation reported to date. Indeed, the concentration of methoxychlor in the water was similar to that which must result from aerial sprays. With such a concentration it seems improbable that simple contact poisoning was responsible for the high mortality observed. Also, the low turbidity of the water (21 JTU) and the small amounts of suspended material (1.5 to 3.6 mg/litre) would probably not have provided sufficient particles containing adsorbed methoxychlor to have poisoned the larvae.

266

RON R. WALLACE,H. B. N. HYNES, W. F. MERRITT

It is possible that the Rhodamine B, which was added with the methoxychlor, may have contributed to the very efficient accumulation of the pesticide by the larvae. As stated above, quantities of the dye were observed on the cuticle and in the gut of larvae caught after the treatment. Wang et al. (1972) found that Rhodamine B greatly increased the adsorption of another insecticide (Parathion) onto clay minerals in water. The mechanism was considered to occur in two steps; first, the Rhodamine B is adsorbed onto the clay surface and Parathion is then associated with the organoclay surface. If the same applies to methoxychlor the high mortality in the Chalk River is understandable. The sizes of larvae drifting in the Chalk River after the treatment generally increased with time, as did the residues of methoxychlor, which suggests that larger larvae are less susceptible and thus accumulate more of the poison than smaller ones. Such accumulation in our laboratory experiments with the particulate formulation indicated that less pesticide was present after 30 min of exposure than after 15 min (Fig. 7). It is possible that this apparently anomalous finding was because the larvae rapidly accumulated a high dose which caused them to stop feeding. Then enzymatic degradation or elimination of the chemical from the larvae may have caused the lower values at 30 min. That this did not occur with the ethanol forfnulation may be because the concentrations of methoxychlor attained were not sufficient to inhibit further uptake of the chemical by the larvae. Other studies have indicated the possibility of recovery from pesticides by blackfly larvae (Travis & Wilton, 1965). The potential for accumulation of methoxychlor in stream invertebrates other than Simuliidae is shown by the residues found in Trichoptera after laboratory exposures (Fig. 7). G. M. Kruzynski (pers. comm.) also found levels in stream invertebrates which ranged from 1-0 to 1.42 mg/kg after laboratory exposures to 0.075mg/litre methoxychlor for 15min. Our experiments indicate that accumulations in Trichoptera were greater with the ethanol formulation (1453-1563/~g/kg) than with the particulate one (615-782#g/kg). This may be caused by adsorption of the former through the large gill surface area of the larvae. The presence of methoxychlor in Trichoptera larvae exposed to a particulate formulation in our experiments indicates that species other than blackfly larvae are susceptible to particulate insecticides. Indeed, Helson (1972) found that particulate methoxychlor used in experimental larviciding of streams severely harmed larvae of Philopotamidae and some types of Chironomidae, as well as Baetidae and Heptageniidae. Clearly, therefore, particulate formulations are no chemical panacea for the selective elimination of Simuliidae from running waters. Residues o f methoxychlor in the Chalk River Burdick et aL (1968) showed that residues of methoxychlor were not detectable

after only 36 days in a lentic ecosystem. Our experiment in the Chalk River, however, indicates that stream vegetation may rapidly accumulate methoxychlor, and that,

EXPERIMENTS WITH METHOXYCHLOR AND SIMULIUM

267

despite some variability in our results, considerable amounts remained even after 8 weeks (Fig. 6). This is possibly enhanced for particulate or oily formulations which may become entangled in vegetation. However, in view of the general declining trend shown by our results and the information on methoxychlor degradation from Metcalf et al. ( 1971) (who described methoxychlor as '... a persistent, biodegradable pesticide...'), it seems highly unlikely that its persistence approaches that of DDT and its isomers in similar situations (Hinden & Bennett, 1970). Merna et al. (1972) found that the rate of breakdown of methoxychlor in water varied considerably with the water source. The half-life in distilled water exceeded 200 days, but it could be as short as 1 day in water with high biological activity. As it is so rapidly and highly concentrated by blackfly larvae, acute poisoning could rapidly extend to predacious invertebrates and fishes in streams. Such a mechanism of concentration was certainly operative in the fish kill in Labrador after D D T larviciding (Hatfield, 1969). Methoxychlor is definitely retained in fish, as shown by Kruzynski & Leduc (1972), but their study may not be directly applicable to the situation in the field after blackfly larviciding, because of the long time (up to 40 days) during which the fish (S fontinalis) were fed contaminated food. Our studies indicate that the catastrophic drift may occur over a much shorter period, but in such a situation fish feeding on contaminated drift may be rapidly exposed to very large doses of methoxychlor. Kapoor et al. (1970) noted that in a model ecosystem methoxychlor was found in fish (Gambusia affinis) at a level 1500 times that of the water. This was much less than that found for comparative studies using DDT. The authors noted that there was considerable evidence that 'Methoxychlor in fish is in a dynamic equilibrium rather than a storage state as with DDT.' However, comparisons with field situations are limited, as the temperatures used in the model were higher than those found in Canadian blackfly streams. Also, discussions of possible toxic effects on fish as a result of methoxychlor larviciding are difficult as many studies often state values as TL mvalues for exposure periods ranging up to 96 h (F,aust, 1964; Merna et al., 1972). However, Merna el al. (1972) noted that the 96 h TL 50 for perch is about 20/~g/litre and indicated that there is a very low tolerance level below which perch are able to metabolise methoxychlor with no mortality. Waiwood & Johansen (1974) found that at 0-1 mg/litre methoxychlor increased oxygen consumption and activity but all the test fish (Catostomus c o m m e r s o m ) died within 85 h. The mechanism of accumulation of methoxychlor in invertebrates is certainly complex. Metcalf et al. (1971) found that in a model ecosystem it was readily metabolised to mono- and di-OHderivatives and was stored at much lower levels than was DDT. However, contrary to the findings of Burdick et al. (1968), Metcalf et al. (197 l) showed that the snail Physa stored methoxychlor to substantial levels and was apparently unable to metabolise it rapidly. This difference in results is most probably due to metabolic differences between different snail species used in the various studies and emphasises the necessity for further experiments in lotic, as well

268

RON R. WALLACE, H. B. N. HYNES, W. F. MERRITT

as lentic, ecosystems as well as comparative work at the specific level for several invertebrates. This discussion therefore indicates that while, in many ways, methoxychlor is much more acceptable as a blackfly larvicide than is DDT, it still presents problems. Even particulate formulations which have sometimes played the role of a 'selective bullet' in some discussions, have their disadvantages. Moreover, the use of dyes as tracers for oil-based insecticide plumes, although of considerable value in field work, appears less satisfactory, in some cases, than had been previously thought. There do seem to be possibilities in the use of enhancers, possibly like Rhodamine B in our experiment, which may make the insecticide more effective in poisoning blackflies. ACKNOWLEDGEMENTS

The authors would like to thank Drs N. Kaushik, P. Corbet, C. Fernando, J. Morton and A. Kempton for their helpful comments in the course of these studies. Drs J. G. Smith and J. Matthews provided invaluable advice and assistance during our laboratory experiments. Dr R. Frank and H. Braun of the Ontario Provincial Pesticide Testing Laboratory at Guelph, Ontario, provided us with the details of techniques for cleanup, extraction and chromatography of the pesticide. Ms N. Williams and D. Pisarczyk drew figures and picked innumerable samples. Drs M. Lock, G. Ware and Ms W. M. Wallace provided vigorous and helpful comments. Dr D. Lush assisted with the coulter counter work. Drs R. Ireland and D. Davies assisted in the identification of biota. The assistance of Cyanamid of Canada Ltd through Mr B. Volkers, and DuPont Canada Ltd through Mr J. Appleton is gratefully acknowledged. The financial support of NRC of Canada is gratefully acknowledged. REFERENCES BURDICK,G. E., DEAN, H. J., HARRIS, E. J., SKEA, J., FRISA, C. & SWEENEY,C. (1968). Methoxychlor as a blackfly larvicide, persistence of its residues in fish and its effects on stream arthropods. N. Y. Fish Game J., 15, 120-42. CHANCE, M. M. (1970). A review of chemical control methods for blackfly larvae (Diptera: Simuliidae). Quaest. ent., 6, 287-92. COLLINS, D. L., TRAVIS,B. C. & JAMNBACK,H. (1952). The application of larvicide by airplane for control of blackflies (Simuliidae). Mosquito News, 12, 75-7. FAUST,S. D. (1964). Pollution of the water environment by organic pesticides. Clin. Pharmacol. Therap., 5, 677-86. FREDEEN, F. J. H. (1974). Tests with single injections of methoxychlor blackfly (Diptera: Simuliidae) larvicides in large rivers. Can. Ent., 106, 285-305. FREDEEN, F. J. H., ARNASON,A. P. & BERCK, B. (1953a). Adsorption of DDT on suspended solids in river water and its role in blackfly control. Nature, Lond., 171,700-1, FREDEEN, F. J. H., ARNASON,A. P., BERCK,B. & REMPEL,J. G. (1953b). Further experiments with DDT in the control of Simulium arcticum Mall. in the North and South Saskatchewan Rivers. Can. J. agric. Sci., 33, 379-93. GJULLIN, C. M., CROSS, F. & APPLEWHITE, H. (1950). Tests with DDT to control blackfly larvae in Alaskan streams. J. econ. Ent., 43, 696-7.

EXPERIMENTS WITH METHOXYCHLOR AND S I M U L I U M

269

HATFIELD, C. H. (1969). Effects of DDT larviciding on aquatic fauna of Bobby's Brook, Labrador. Can. Fish Cult., 40, 61-72. HELSON, B. V. (1972). The selective effects of particulate fo rmulations of insecticides on stream fauna when applied as blackfly (Diptera: Simuliidae) larvicides. M.Sc. Thesis, Queens University, Kingston, Ontario. HINDEN, E. & BENNETT, P. J. (1970). Transport of organic pesticides to the aquatic environment. Adv. Wat. Pollut. Res., 2. HOWMILLER, R. P. (1972). Effects of preservatives on weights of some common macrobenthic invertebrates. Trans. Am. Fish. Soc., 101,743~. JAMNBACK,H. (1973). Recent developments in control of blackflies. A. Rev. Ent., 18, 281-304. JAMNBACK,H. • FREMPONG-BOADU,J. (1966). Testing blackfly larvicides in the laboratory and in streams. Bull WId Hlth Org., 34, 405-21. JENSEN, L. n. & GAUFIN,A. R. (1964). Effects often organic insecticides on two species of stonefly naiads. Trans. Am. Fish. Soc., 93, 27-34. KAPOOR, I. P., METCALF, R. L., NYSTROM, R. F. & SANGHA, G. K. (1970). Comparative metabolism of methoxychlor, methiochlor and DDT in mouse, insects, and in a model ecosystem. J. agric. Fd Chem., 18, 1145-52. KENNEDY, H. D., ELLER, L. L & WALSH, D. F. (1970). Chronic effects of methoxychlor on Bluegills and aquatic invertebrates. Tech. Pap. Bur. Sport Fish. Wildl. U.S. Dep. lnt,, 53, 17pp. KERSHAW, W. E., WILLIAMS,T. R., FROST, S. & HYNES, H. B. N. (1965). Selective effect of particulate insecticides on Simulium among stream fauna. Nature, Lond., 208, 199. KERSHAW, W. E., WILLIAMS,T. R., FROST, S., MATCHETT,R. E., MILLS, M. L. & JOHNSON, R. n. (1968). Trans. R. Soc. trop. Med. Hyg., 62, 35-40. KRUZYNSKI, G. M. & LEDUC, G. (1972). Methoxychlor, a new threat to the Atlantic salmon. Atlant. Salmon J., 1, 5pp. LANGOIS, B. E., STEMP, A. R. & LISKA, B. J. (1964). Analysis of animal food products for chlorinated insecticide residues. Column cleanup of samples for electron' capture gas chromatographic analysis. J. Milk Fd Technol., 27, 202-4. MCLAY, C. (1970). A theory concerning the distance travelled by animals entering the drift of a stream. J. Fish. Res. Bd Can., 27, 35~70. MACLEOD, H. A., WALES,P. J., GRAHAM,R. A., OSADCHUK, M. & BLUMAN,N. (eds) (1969). Analytical methods for pesticide residues in foods. Oftawa, Ontario, Queen's Printer. MERNA, J. W., BENDER,M. E. & N ovY, J. R. (1972). The effects ofmethoxychlor on fishes. I. Acute toxicity and breakdown studies. Trans. Am. Fish. Soc., 101,298-301. METCALL R. L., SANGHA,G. K. & KAPOOR, I. E. (1971). Model ecosystem for the evaluation of pesticide biodegradability and ecological magnification. Environ. Sci. & TechnoL, 5, 709-13. MUIRHEAD-THOMPSON, R. C. (1971). Pesticides and freshwater fauna. New York, Academic Press. N OEL-BUXTON, M. B. (1956). Field experiments with DDT in association with finely divided inorganic material for the destruction of the immature stages of the genus Simulium in the Gold Coast. Jl W. Afr. Sci. Ass., 2, 36-40. PETERSON, B. V. & DAVIES, D. M. (1960). Observations on some insect predators of blackflies (Diptera: Simuliidae) of Algonquin Park, On.tario. Can. J. Zool., 36, 9 18. POWER, J. (1966). Observations of the speckled trout (Salve~inusfontinalus) in Ungava. Naturaliste van., 93, 187-99. STRICKLAND, A. H. (1954). An aphid counting grid. PL Path., 3, 73-5. TRAVIS, B. V. & WILTON, D. P. (1965). A progress report on simulated stream tests of blackfly larvicides. Mosquito News, 25, 112-18. WA1WOOD, K. G. & JOHANSEN, P. H. (1974). Oxygen consumption and activity of the white sucker (Catostomus commersonii) in the lethal and non-lethal levels of the organochlorine insecticide methoxychlor. Wat. Res., 8, 401-6. WALLACE, R. R. (1971). The effects of several insecticides on blackfly larvae and on other stream-dwelling aquatic invertebrates. M.Sc. Thesis, Queen's University, Kingston, Ontario. WALLACE, R. R. (1973). The effect of methoxychlor (1,1,1-trichloro-2, 2-bis (p-methoxyphenyl) ethane) on, and the accumulation of methoxychlor in, some insects of running waters. Ph.D. Thesis, University of Waterloo, Waterloo, Ontario. WALLACE,R. R., MERRITT, W. F. & WEST, A. S. (1973a). Dispersion and transport of Rhodamine B dye and Methoxychlor in running water: A preliminary study. Environ. Pollut., 5, 11 18. WALLACE,R. R., WEST, A. S., DOWNE, A. & HYNES, H. B. N. (1973b). The effects of experimental blackfly (Diptera: Simuliidae) larviciding with abate, dursban and methoxychlor on stream invertebrates. Can. Ent., 105, 817-31. WANG, W., LEE, G. F. & SPYRIDAKIS,D. (1972). Adsorption of parathion in a multicomponent solution. Wat. Res., 6, 1219-28.