Ptstic'. S C ~ . 1994, 41, 339-343

Non-Invasive Method for Monitoring the Exposure of Barn Owls to Second-Generation Rgdenticides Alan Gray,* Charles V. Eadsforth, Alan J. Dutton Shell Research Limited, Sittingbourne Research Centre, Sittingbourne, Kent, ME9 8AG, UK

& John A. Vaughan Central Science Laboratory, MAFF, Worplesdon, Surrey, GU3 3LQ, UK

(Revised manuscript received 27 January 1994; accepted 26 April 1994))

Abstract: Chemical analysis of rodenticide residues in regurgitated owl pellets has been shown to be a sensitive, non-invasive method for monitoring the potential exposure of Barn Owls to second-generation rodenticides in their prey. The method, originally developed for flocoumafen, has been extended to two other rodenticides, brodifacoum and difenacoum.

The method was validated as part of a toxicity study in which Barn Owls were separately fed 40-130 pg day- I of brodifacoum, difenacoum and flocoumafen in mice over 15 days. Each day an average of 25% of the consumed rodenticide was regurgitated in the pellets.

1 INTRODUCTION

The environmental risk of rodenticide use depends not only on the toxicity of the rodenticide, but also on the exposure of the non-target animal. In recent years there has been some concern regarding the potential for secondary poisoning of predators and scavengers from anti-coagulant rodenticide baits used on farms. The Barn Owl (Tyro alba Scop.) is a night-hunting predator which feeds on live rodents and birds' and might therefore be considered potentially at risk .from residues of rodenti- cides in its prey.

The chemical analysis of rodenticide residues in regurgitated Barn Owl pellets has been developed as a sensitive non-invasive method for monitoring the exposure of the owls to flocoumafen in their prey. This method was validated with a single-dose study employing radiolabelled flocoumafen,2 and also as part of a 1 -, 3- and 6-day toxicity feeding study,3 where owls were fed mice which had previously been fed flocoumafen bait. In these studies, respectively 15% and 22-32% (mean 27%) of the

* To whom correspondence should be addressed.

Pestic. Sci. 0031-613X/94/$09.00 $'> 1994 Shell Research Ltd 339

daily consumed flocoumafen was regurgitated in the owl pellets during the 24 hours following feeding.

This paper reports an extension of the method to two other second-generation rodenticides, brodifacoum and difenacoum, and presents additional validation data for flocoumafen. The data were obtained as part of a toxicity feeding study4 in which Barn Owls were fed for a period of 15 days on mice which had previously been fed separately on brodifacoum, difenacoum or flocoumafen 0.05 g kg-' wax block bait.

2 MATERIALS AND METHODS

2.1 Chemicals

The three rodenticides employed were:

Brodifacoum; 3-[3-(4'- bromo biphenyl-4-y1)- 1,2,3,4- tetrahydro- 1 -naphthyl]-4-hydroxycoumarin.

Difenacoum; 3-(3-biphenyl-4-yl-1,2,3,4-tetrahydro- 1 -naphthyl)-4-hydroxycoumarin.

Flocoumafen; 4-hydroxy-3-[ 1,2,3,4-tetrahydro-3- [4-(4-trifluoromethylbenzyloxy)phenyl]- 1 - naph t h yl]-coumarin.

340 Alan Gray, Charles V. Eadsforth, Alan J . Dutton, John A. Vauyhan

2. I. 1 Rollenticide bait Three commercial wax block baits were employed. Each nominally contained 0.05 g kg- of the rodenti- cide, which was confirmed by analysis. These were:

Brodifacoum, ‘Klerat’* bait (Ref No. ST90/395); Difenacoum, ‘Ratak’* bait (Ref No. ST90/396); Flocoumaren, ‘Storm’* bait (Ref No. ST90/388).

2.1.2 Authentic standards Analytical reference (calibrated technical) material of the three rodenticides was obtained as follows:

brodifacoum, source Sorex Limited, ref 1 /88, purity 97.9% (w/w), cis:trans isomer ratio 2.3: 1;

difenacoum, source Sorex Limited, ref 100 H/2/3/4, purity 99.5% (w/w), cis:trans isomer ratio 3.3: I ; and

flocoumafen, source Sittingbourne Research Centre, ref 2104001/90, purity 97.8% w/w, cis:trans isomer ratio 1.2: 1.

2.2 Animal experiments

2.2.1 Mice feeding phase Male mice, strain Harlan Olac Hsd/Ola:ICR, age 4-6 weeks and nominal weight range 20-25 g supplied by Harlan Olac Ltd, Bicester, England were employed in batches as required.

After acclimatisation, batches of mice were either:

( i ) housed singly and allowed to feed on weighed amounts of bait (between 1.0 g and 5.0 g) for 22 or 23 h followed by 4-5 h on undosed Laboratory Animal Diet (supplied by Special Diet Services, Witham, Essex) prior to killing, or

(ii) housed in groups of 20 and fed ad libitum for either 24 or 48 h.

The mice were then killed with carbon dioxide, individu- ally weighed, packaged, and stored at c. -18°C until required. The weight of the mice after treatment ranged from 20 to 34 g, mean 26 g.

Representative numbers of mice (average five mice per batch) from individual batches were analysed by a method4 closely related to that used for the pellets (Section 2.3) to determine the mean rodenticide present in the separate batches.

2.2.2 0wl.feediny phase A total of twelve captive-bred Barn Owls of mixed sexes, weighing 320-455 g during the treatment phase were used. They were individually housed in outdoor flight pens (4.5 m x 2 m x 2 m) constructed from wire netting on a wooden frame with a base of concrete slabs and

* ‘Klerat’ and ‘Ratak’ are Zeneca Lid trademarks, and ‘Storm’ is an American Cyanamid trademark.

fitted with partial shelter, nest box, perch and water bowl. Prior to treatment they were acclimatised for 15 days on a daily ration of up to four untreated mice (c. 80 g day-’ bird- ’).

The owls were fed for 15 days in groups of three, one bird per r~dent ic ide .~ The amount of rodenticide offered was adjusted by varying the numbers of treated mice offered and their residual rodenticide content. The balance of the feed was made up with untreated mice, and water was offered ad libitum. The net rodenticide consumption of each owl was measured by analysing the food remains and subtracting any rodenticide present from the amount offered.

Owls surviving the 15-day treatment were fed on untreated mice for a further 15 days or until they died.

Regurgitated owl pellets were collected daily during the acclimatisation, treatment and post-treatment periods and stored deep-frozen (c. -1SOC) prior to analysis. Those pellets collected during the acclimatisation period were used as analysis control samples.

2.3 Pellet analysis method

The pellets were extracted by homogenisation in acetone + chloroform (1 + 1 by volume) and anhydrous sodium sulfate. Mixtures were filtered and evaporated to incipient dryness in a stream of dry nitrogen. The resulting residues were redissolved in methyl tert-butyl ether and cleaned up using pre-washed Bond Elut-NH, cartridges, eluting with methyl tert-butyl ether + acetic acid (9 + 1 by volume). The flocoumafen concentrations in the sample extracts were quantified by high performance liquid chromatography (HPLC) by comparison with reference standards of the three rodenticides, each isomer being separately determined.

A Pye Unicam PU 4010 pump and Perkin Elmer ISS 100 autosampler fitted with a 100-pl loop injector were employed in conjunction with a Perkin Elmer LS1 fluorescence detector with excitation wavelength 3 10 nm and emission wavelength 390 nm. An S5 ODs-2 (5-pm) column (Hichrom, Reading, 25 cm x 4.6 mm i.d) was eluted with acetonitrile+ water+ acetic acid, (80+20+01 by volume) at a flow rate of 2.0 ml min- ’. Post-column methanol + triethylamine (80 + 20 by volume, 0.2 ml min-’) was added via a mixing connector ‘T’ to increase the pH of the eluent to over 8 and so improve fluorescence sensitivity. The following mean retention times were obtained: brodifacoum: cis 5.7 min, trans 6.4 min; difenacoum: cis 4.0 min, trans 4.6 min; and flocoumafen: cis 4.4 min, trans 5.2 min.

In this study it was not a requirement to resolve chromatographically all three rodenticides and their individual cis: trans isomers. However, this may be achieved using the same chromatographic conditions but with two of the HPLC columns connected in series. Retention times obtained under these conditions were brodifacoum: cis 17.0 min, trans 18.7 min; difenacoum:

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342 Alan Gray, Charles V. Eadsforth, Alan J . Dutton, John A . Vauyhan

TABLE 2 Rodenticide Content of Pellets Regurgitated during Treatment Period-Summarised Data

Brodifacoum

Owl No. __

Difenacoum

Owl No. Owl N o . - ~ - ~ ~-~ -~ ~ __

I2 2 9 1 8 4 3 7 / I ~ - - - ~- ~ ~- - _ _ - - ~-

Average amount of rodenticide consumed ( p g day I ) 52 99 133 50 101 128 39 43 85

- - -~ _ _ - - ~ ~~

Average amount rodenticide regurgitated in pellets (!ig day ~ ' ) 19 27 32 10 31 35 5.5 10 21

- ~- - . - _ - - ~ ~

Average rodenticide regurgitated in pellets (7;J 37 27 24

brodifacoum mean = 29%

20 31 27 difenacoum mean = 26%

14 23 25 fl oco u m afe n mean = 21%

Overall mean weight of the owls during treatment = 380 g. Pellet samples from an additional three owls, Nos. 5 , 6 and 10, were not analysed. Owls No. 4, 6, 7 and 9 died after 15 days' treatment f 1 day.

cis 10.6 min, truns 1 1.8 min; and flocoumafen: cis 12.4 min, trans 14.6 min.

The method was validated by analysis of control pellets (obtained during the acclimatisation period) and control pellets fortified separately with the three rodenticides. The control pellet samples contained less than 0.01 mg k g - ' ofeach rodenticide (i.e. on an average weight of 5 g day- pellet, <0.05 pg day-'). With fortified control pellets, mean recoveries of 79% ( & 15), 99% (_+ 19) and 957; (_+ 7) were obtained respectively with brodifacoum, difenacoum and flocoumafen.

3 RESULTS

3.1 Owl feeding phase

Each owl was fed one of the three rodenticides, brodifa- coum, difenacoum or flocoumafen, in mice in amounts which ranged from 50 to 220 pg day- '. Considerable amounts were rejected, up to 40% with brodifacoum and flocoumafen, and up to 70% with difenacoum. Thus the actual amount consumed by the owls was considerably reduced to: brodifacoum 52- 133 pg day-'; difenacoum 36-128 pg day-'; and flocoumafen 39-85 pg day-'. These are equivalent to 0.13-0.39, 0.10-0.37 and 0.12- 0.19 mg kg- ' owl body weight per day for the three rodenticides, respectively (Tables 1 and 2) .

Four of the owls died after 15 days' treatment i 1 day, one each after treatment with brodifacoum and difenacoum, and two with flocoumafen. These owls had consumed

amounts of rodenticide equivalent to 0.39, 028,0.15 and 0.19 mg kg-' owl body weight per day, respectively.

3.2 Rodenticide residues in owl pellets

The individual rodenticide contents in regurgitated owl pellets collected during the treatment and post-treatment periods are presented in Table 1. Because of the large number of samples, a representative proportion of the total was analysed and in addition certain of the pellets collected during the treatment period were analysed in groups of two or three samples bulked together.

The rodenticide contents in pellets collected during the treatment period were:

brodifacoum-range 0.14-55 pg day-',

difenacoum-range 3.7-50 pg day-',

flocoumafen-range 1.6-36 pg day-',

mean 26 pg day

mean 25 pg day'

mean 12pgday- '

The mean proportions of the rodenticides regurgitated in the pellets during the treatment period, expressed as percentages of the amount consumed, were 29%, 26% and 2 1 % respectively for brodifacoum, difenacoum and flocoumafen.

Post-treatment, the levels of rodenticide in the pellets of the surviving owls declined rapidly, such that, by day three post-treatment, the residues were reduced to 6 10% of the mean value during treatment. At the end of the 15-day post-treatment period, the levels of ail three

Monitoring exposure qf barn owls to rodenticides 343

rodenticides had declined to about 1 % of the level during treatment.

4 DISCUSSION

The residues of brodifacoum, difenacoum and flocoumafen in pellets collected during the treatment period were very consistent, averaging between 21 and 29% of the amounts consumed. These data are also comparable with flocou- mafen residues present following a single dose study2 with radiolabelled flocoumafen, 15%, and a 1-, 3- and 6-day feeding study,3 27%. In these studies Barn Owls were also fed on mice which had previously consumed flocoumafen. Post-treatment, the levels of all three rodenticides declined rapidly.

The practical application of the method was examined as part of a recent field study5 on the environmental impact of commercial rodenticide use in Eire. Barn Owl roosts were monitored and fresh owl pellets readily collected during a two-week period in Winter/Spring when rodenticide bait was being used by local farmers.

No residues of brodifacoum, difenacoum or flocoumafen were found in pellets, indicating that over the period of study none of the owls was exposed to detectable amounts of the three rodenticides in their prey.

Thus the non-invasive pellet analysis method for monitoring the exposure of owls to rodenticides, originally developed for flocoumafen.2 is also applicable to two other second-generation coumarin-based rodenticides, brodifacoum and difenacoum. On average, approximately 25% of the consumed rodenticide is regurgitated in owl pellets.

REFERENCES

1. Mikkola, H., Owls of Europe, (1983). Pitman Press, Bath. 2. Eadsforth, C. V., Dutton, A. J., Harrison, E. G., & Vaughan,

J. A., Pestic. Sci., 32 (1991) 105-19. 3. Newton, I., Wyllie, I.. Gray, A., & Eadsforth, C. V., Pestic.

Sci., in press. 4. Eadsforth, C. V., Gray, A., Dutton, A. J., & Vaughan, J. A,,

Pestic. Sci. (submitted). 5. Harrison, E. G., Eadsforth, C. V., & Vaughan, J. A,, Brighton

Crop Prot . Con$, Pests and Diseases, 8C-1 (1990) 951--6.