Lethality of Pyrethrins to Larvae and Postlarvae of the American Lobster (Homarus americanus)

ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY ARTICLE NO. ES971571

38, 150–154 (1997)

Lethality of Pyrethrins to Larvae and Postlarvae of the American Lobster (Homarus americanus) L. E. Burridge and K. Haya Marine Environmental Sciences Division, Department of Fisheries and Oceans, Biological Station, St. Andrews, New Brunswick, E0G 2X0, Canada Received April 23, 1997

Pesticide formulations containing pyrethrins are being used to treat salmonids for infestations of the copepod parasites Lepeophtherius salmonis and Caligus elongatus (sea lice). The acute lethality of one such formulation to four larval stages of the American lobster (Homarus americanus), a species of significant economic importance in eastern Canada, was determined. The formulation tested contained 0.06% pyrethrins and 0.6% piperonyl butoxide (a synergist). Stage I larvae (48-h LC50 = 4.42 µg/ liter) were significantly less sensitive than stage II, III, or IV larvae. Stage II larvae (48-h LC50 = 2.72 µg/liter) were significantly less sensitive than Stage III or IV larvae. Stage III and IV larvae were not significantly different in their response to the pyrethrins formulation (48-h LC50 = 1.39 and 0.73 µg/liter, respectively). Most published studies using lobster larvae have reported that the earliest larval stage was the most sensitive to chemicals. The results described here indicate that the earliest larval stage is the least sensitive to the pyrethrins formulation. © 1997 Academic Press

INTRODUCTION

The parasitic copepods Lepeophtheirus salmonis and Caligus elongatus (also referred to as sea lice) have caused significant losses in the aquaculture industry in Europe (see, e.g., Roth et al., 1993). Sea lice are a cause for concern in the salmonid aquaculture industry in Canada as well (Cusack and Johnson, 1990). Severe infestations of sea lice occurred in the Bay of Fundy in the fall of 1994 and in the summer and fall of 1995 and 1996. Sea lice can be present on caged fish throughout the year. In the Bay of Fundy, sea lice are most prevalent during the fall when water temperatures have risen to between 10 and 14°C (Hogans and Trudeau, 1989). A number of methods have been used to control sea lice in Europe. The most widely used method has involved treating fish with organophosphate pesticides (Roth et al., 1993). In Canada, formulations containing hydrogen peroxide, azamethiphos, and pyrethrins have been used under temporary registration for treatment of sea lice infestations of caged Atlantic salmon. Pyrethrins were the first of these active ingredients to be used in Canada to combat sea lice. Their use in Europe has been described by Boxaspen and Holm (1991) and Roth et al. 150 0147-6513/97 $25.00 Copyright © 1997 by Academic Press All rights of reproduction in any form reserved.

(1993). The pyrethrins are extracted from the flower Chrysanthemum cinerariaefolium. The active constituents of the extract are collectively referred to as ‘‘pyrethrins’’ (Davies, 1985). The pyrethrins consist of the individual compounds cinerin I and II, pyrethrin I and II, and jasmolin I and II in varying proportions. One proposed treatment of salmon against sea lice is a 1-h bath in a solution of pyrethrins at a concentration of 10 mg/liter. Initially, a veterinary formulation, Vet Kem Flea and Tick Pump Spray (Zoecon Canada Inc., Whitby, Ontario), was proposed for use and was used in several field trials. The Vet Kem formulation includes pyrethrins (0.06%) and piperonyl butoxide (0.6%). Piperonyl butoxide serves as a synergist added to pyrethrins to increase toxicity by inhibiting detoxification pathways (Roth et al., 1993). More recently, a formulation containing pyrethrins (0.1%) and piperonyl butoxide (1.0%) was granted emergency registration status in Canada. Concentrations of up to 40 mg/liter were applied to cages under this registration. Each formulation is prepared in water from an emulsifiable concentrate. The emulsifiable concentrate contains 3% pyrethrins and 30% piperonyl butoxide. The remainder of the concentrate comprises petroleum solvent, butanol, petroleum distillate, and xylene at concentrations that are considered proprietary information. The approximate amounts of individual pyrethrins that make up the 3% pyrethrins are as follows: cinerin I, 20%; cinerin II, 15–20%; pyrethrin I, 25– 30%; pyrethrin II, 1–2%; jasmolin I, 25–30%; jasmolin II, 1–2%. The American lobster (Homarus americanus) inhabits many of the areas currently used for salmon aquaculture in Eastern Canada. This paper describes a series of laboratory experiments conducted to determine the acute lethality of pyrethrins (Vet Kem formulation) to larvae and postlarvae of the American lobster. MATERIALS AND METHODS

Berried female lobsters (H. americanus) were held at 20°C to induce release and hatching of their eggs. Stage I larvae were collected daily and transferred to plexiglass holding tanks. The tanks were well aerated and the water temperature was maintained at 15 ± 1°C. The larvae were fed live brine shrimp daily. Larvae were collected from several females during the course of the experiments. The larvae were checked daily to determine their stage of development.

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Twenty-four hours prior to beginning the lethality tests the larvae were transferred to a 2-liter glass beaker with aerated seawater maintained at 10 ± 1°C. The larvae were not fed in these containers nor were they fed during the exposure. The sizes of the larvae are as follows:

LT50 was calculated as the geometric mean between the shortest period at which 50% or greater of the animals died and the longest period of time where less than 50% of the exposed animals died. Water was sampled and analyzed at T 4 0 to confirm the presence of pyrethrins.

Stage I 7.0 5 0.8 mg/larva Stage II 10.8 5 0.6 mg/larva Stage III 16.6 5 2.9 mg/larva Stage IV 36.8 5 2.9 mg/larva

Water Analysis

Two sets of experiments were conducted to determine the effects of pyrethrins on larvae. Acute Lethality of Vet Kem Formulation to Larvae Vet Kem Flea and Tick Pump Spray was purchased from a local veterinary clinic. The spray contains 0.06% pyrethrins and 0.6% piperonyl butoxide. Dilutions of the formulation were prepared in 200 ml of seawater in 400-ml glass beakers. Ten larvae were added to each beaker. The solutions were lightly aerated throughout the 48-h exposure period. Control vessels contained seawater and larvae. Initial experiments were conducted with five concentrations and controls (in duplicate). This was later expanded to eight concentrations and controls (duplicate) to more clearly define the lethal threshold. The beakers were checked at 1, 3, 6, 12, 24, and 48 h, and the number of dead at each concentration was recorded. Larvae were considered dead when no evidence of movement was visible even after gentle prodding. In most cases death was easily determined since a color change is noted. The dead larvae were removed from the beakers. During several tests an extra exposure beaker (including larvae) was included to allow for water sampling. Twentymilliliter samples were taken (using a glass volumetric pipet) from this beaker at 0, 1, 3, 6, 12, 24, and 48 h to determine the concentration of pyrethrins. The 48-h LC50 was determined according to Stephan (1977) using the moving average method. The results of tests in which there was greater than 10% mortality of control larvae were not used in any calculations. An analysis of variance was performed on the data. The significance of differences between mean LC50’s for each stage was determined using a t test (Sokal and Rohlf, 1969). Effect of 1-h Exposure on Lobster Larvae The protocol for treating fish for sea lice includes a 1-h bath in 10 mg/liter pyrethrins. To determine if a 1-h exposure to the Vet Kem formulation followed by transfer to clean seawater would have any effect on the larvae, they were exposed to two concentrations of pyrethrins, one known to be toxic during the acute lethality tests and 10 mg/liter (the treatment concentration). In one set of beakers the exposure proceeded as previously described; in another set the larvae were transferred to beakers containing ‘‘clean’’ seawater after 1 h. Where deaths occurred the time to 50% mortality (LT50) was calculated. The

To the 20-ml sample of water 20 ng of phenanthrene (in ethanol) was added to serve as an internal standard for the analytical procedure. The water was then extracted by gentle mixing on a magnetic stirrer with 2 ml of hexane for 30 min. The hexane layer was removed, placed in a round-bottom flask, and reduced to almost zero volume under vacuum. The sample was then brought to volume (100 or 200 ml) in hexane spiked with 200 ng/ml hexachorobenzene (HCB). The sample was analyzed by gas chromatography–mass spectrometry (GC/MS) on a Finnigan 4500 GC/MS/DS. The HCB serves as an instrumentation standard for gas chromatograph/mass spectrometer. The GC oven was programmed as follows: 60°C for 2.5 min, to 180°C at 20°C/min, to 250°C at 4°C/min, and held at 250°C for 10 min. The injector temperature was 250°C. A Supelco PTE-5 30-m capillary column (0.25-mm i.d.) with a 0.25-mm film was used to separate the compounds. The column effluent was analyzed by electron impact (EI) ionization in the MID range. Seven ions, m/z 4 107.1, 123.2, 133.2, 163.2, 178.2, 283.8, and 338.1, were monitored at a 393-ms sample time. The concentration of pyrethrins was determined by comparison to standard solutions of the compounds. The commercially available pyrethrum extract used to prepare standards contained only 25% pyrethrins. The formulation tested was guaranteed to contain 0.06% pyrethrins, but it was unclear whether this means 0.06% of the actual pyrethrins or 0.06% of pyrethrum extract (which may be only 25% pyrethrins). The CAS number for active ingredient in the formulation is that of pyrethrin I, thus adding to the confusion. Analyses indicated that all six compounds were present and calculations were made assuming that the formulation was 0.06% pyrethrins. The pyrethrins were quantified using cinerin I. The peak for cinerin I has the largest area using the previously described GC/MS conditions. The concentration of this compound decreased in an exponential fashion. The constants that describe this exponential decrease were used in conjunction with the initial nominal concentration of pyrethrins to calculate the average concentration during the exposures according to Zitko et al. (1977). RESULTS

Cinerin I decreased exponentially during a typical lethality test (Fig. 1). The formula in Fig. 1 was used to determine average nominal concentrations. The calculated LC50 (48 h) of the pyrethrins formulation to the four larval stages of lobster are listed in Table 1. The concentrations reported are those for total pyrethrins (assuming that all decreased with time in the same fashion as cinerin I). Analysis of variance indicated that there were significant

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FIG. 1. Disappearance of cinerin I from water in a 48-h lethality test. The formula for the exponential decrease is in the upper right corner.

differences among the LC50’s (P < 0.05). Stage I larvae (48-h LC50 4 4.42 mg/liter) were significantly less sensitive to the Vet Kem formulation than were stage II, III, or IV larvae (t test, P < 0.05). Stage II larvae (48-h LC50 4 2.72 mg/liter) were significantly less sensitive than stage III and IV larvae (48-h LC50 4 1.39 and 0.73, respectively, t test, P < 0.05). Stage III and IV larvae were not significantly different in their sensitivity to pyrethrins. The results of the experiments in which larvae were either left in the treated solution or transferred to ‘‘clean’’ water after 1 h are listed in Table 2. Transfer of the larvae to ‘‘clean’’ water reduced the number of mortalities over the 48 h and increased the LT50. The mortalities were significantly greater in all treated groups compared with the controls. DISCUSSION

There are only a few examples in the literature of studies in which the relative sensitivity of lobster larvae to xenobiotics or TABLE 1 48-h LC50 of Pyrethrins (µg/liter) to Larval Stages of the American Lobster (Homarus avericanus) Lobster stage

n

LC50a (mg/liter)

Range

I II III IV

7 6 6 5

4.42 ± 0.84 2.72 ± 0.43 1.39 ± 1.02 1.02 ± 0.31

3.72–6.03 2.13–3.38 0.52–3.13 0.39–1.06

a

Mean ± SD.

environmental variables is compared. Wells and Sprague (1976) report that the 96-h LC50 of crude oil to lobster (H. americanus) larvae increases with larval stage (0.86 mg/liter at stage I to 4.9 mg/liter at stages III and IV). Wells (1972) reports that stage I to stage III lobster larvae are more sensitive to Venezuelan crude oil than is stage IV. Capuzzo et al. (1984) found that the respiration rate of larval H. americanus dropped in response to exposure to Louisiana crude oil. Stages I and II reacted more quickly than did later stages. Fisher et al. (1976) exposed H. americanus larvae to malachite green and found no difference in sensitivity between larval stages. Xenobiotics tend to reduce growth and extend the period in larval lobsters (Wells, 1972; Fisher et al., 1976; Wells and Sprague, 1976; Capuzzo et al., 1984). Present studies indicated that the later larval and postlarval stages are more sensitive to pyrethrins than the earlier stages. Several authors determined the sensitivity of larvae to environmental stressors. Young-Lai et al. (1991) tested stages I to III larvae and stage IV postlarvae of H. americanus with ammonia and found each successive larval stage was more tolerant of the ammonia than the previous. Gruffydd et al. (1975) found older larvae (H. gammarus and H. americanus) to be more tolerant of elevated water temperatures, especially over short periods, than the younger larvae. Stage II larvae, however, were more sensitive than any other stage. This finding is confirmed by Sastry and Vargo (1977). In fact, they found stage II larvae had a lower temperature tolerance than any other crustacean larvae tested. Charmantier et al. (1988) looked at salinity tolerance in H. americanus and Penaeus japonicus and found that, in H. americanus, salinity tolerance was greater in

LETHALITY OF PYRETHRINS TO AMERICAN LOBSTER

TABLE 2 Comparison of Percentage Mortality and LT50 of Lobster (Homarus americanus) Larvae Exposed to Pyrethrins Formulation for 48 and 1 h

Treatment

Pyrethrins concentration (mg/liter)

Mortality (%) at 48 h

LT50 (h)

I I I I I

Control 48 h 1h 48 h 1h

0.0 9.6 9.6 84 84

0 20 0 93 80

17 34

II II II II II

Control 48 h 1h 48 h 1h

0.0 9.6 9.6 84 84

10 28 50 100 100

34 34 34

III III III III III

Control 48 h 1h 48 h 1h

0.0 9.6 9.6 42 42

5 77 37 100 73

IV IV IV IV IV

Control 48 h 1h 48 h 1h

0.0 9.6 9.6 42 42

10 100 35 100 100

Lobster stage

34 17 34 25 17 12

stage I than in stage II, greater in stage II than in stage III, and greater in stage IV than in any other stage. They also noted that salinity tolerance decreased near the time of molting. In general, larvae are more sensitive to chemicals than adults (Conner, 1972; Johnson and Gentile, 1979; Young-Lai et al., 1991). Johnson and Gentile (1979) even suggested setting regulatory limits on some metals based on larval testing since this should provide adequate protection for (less sensitive) adults. Early postlarval lobsters (i.e., stage IV) would not be adequately protected from the effects of pyrethrins if regulatory limits were set according to larval (i.e., stages I to III) toxicity. The 48-h LC50’s reported here are well below the treatment concentration (10 mg/liter) used to control sea lice on salmonids. In the present study, stage II and III lobster larvae and stage IV postlarvae were killed after only 1 h exposure to this treatment concentration. No attempt was made to determine concentrations at which no effect was evident. During most tests, deaths were observed at all concentrations. It is difficult to attribute all deaths to the pyrethrins. Wells and Sprague (1976) have identified cannabalism as a source of mortality when dealing with larval lobsters. Some cannibalism was observed during the current tests, and partially eaten carcasses were seen in the control vessels during some tests. The larvae in control vessels and those exposed to low concentrations of pyrethrins remained very active throughout the testing. At higher pyrethrin concentra-

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tions the larvae became moribund soon after initiation of the tests. At the higher concentrations the level of activity was low; there was no evidence of cannibalism and carcasses of dead larvae were not eaten by those still living. Fisher et al. (1976) reported that the appendages of larvae were lost during shortterm exposure to high concentrations of malachite green. McHenery et al. (1991) also reported occasional loss of claws in larvae of the European lobster (H. gammarus L.) exposed to dichlorvos. This phenomenon was observed with stage IV postlarvae. The claws seem to fall off during the first 2 h of exposure to pyrethrins at high concentrations. Unlike in the findings of Fisher et al., these larvae were not necessarily the ones that later died, i.e., mortality was observed in larvae with or without claws. Some lobsters survived even after losing their claws. The risk of adverse effects on the $7 million Bay of Fundy lobster industry from pyrethrins is dependent not only on the concentration at which toxic effects are manifested, but also on the likelihood that lobsters experience exposure to toxic concentrations. The American lobster (H. americanus) inhabits many of the areas currently used for salmon aquaculture in Eastern Canada. Also, the concentration of pyrethrins that can be lethal to larval lobster is below the concentration recommended for treatment of sea lice. Hatching of eggs and development of larvae are temperature dependent and usually occur in late June and early July (Phillips and Sastry, 1980). In the Bay of Fundy, where the water temperature usually reaches a maximum of 14°C early in the fall, larval production can continue through August and has been seen in September (YoungLai personal communication). The larval stages (I–III) of the lobster are pelagic. The first postlarval stage (stage IV) spends at least some of its time in the water column prior to settling to the bottom (Charmantier et al., 1991). It is possible that treatment of sea louse-infested fish could coincide with the presence of lobster larvae in the water. Indeed, it is possible that the most sensitive stage (IV) may be most abundant when the sea lice are also abundant. CONCLUSION

The results of laboratory studies obviously cannot be taken as proof of what will occur in the field. Pyrethrins degrade quickly in sunlight (Leahey, 1985). Greater than 30% loss of pyrethrins from seawater has been noted after 1 h exposure to sunlight. The same level of degradation is not seen under laboratory lighting (Zitko, Burridge, and Haya, unpublished results). There are certainly other environmental factors that may mitigate the effects of pyrethrins. Subsequent to performing these experiments, limited field trials using pyrethrins have been conducted in the Bay of Fundy. Lobster larvae situated around the treated cage revealed no adverse effects when the chemical was released (Hogans, O’Halloran, and Burridge, unpublished results). Field testing of antilouse treatments is expected to continue in the Bay of Fundy. It is recommended that

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lobsters (particularly stage IV) should be used as sentinels during these studies. As well, the difference in sensitivity of the various larval stages would indicate that further studies of this nature should be conducted with any chemical that may be used to treat salmon against sea lice infestations. ACKNOWLEDGMENTS The authors thank Ms. Susan Waddy and Mr. Wilfred Young-Lai for providing the lobster larvae. Hugh Akagi, Marcel Babineau, and Ken MacKeigan performed the water analyses. W. E. Hogans, Dr. V. Zitko, and Dr. R. L. Saunders provided valuable comments on the manuscript.

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