Risk Assessment for Honeybees from Pesticide-Exposed Pollen

Ecotoxicology, 9, 287]297, 2000Q 2000 Kluwer Academic Publishers. Manufactured in The Netherlands.

Risk Assessment for Honeybees from Pesticide-Exposed Pollen

SARA VILLA,1 MARCO VIGHI,*,1 ANTONIO FINIZIO,1

GRAZIELLA BOLCHI SERINI2

1 Department of Enïronmental and Landscape Science, Uni ersity of Milano Bicocca,Milano Italy, Piazza della Scienza 1, 20126 Milano Italy

2 Institute of Agricultural Entomology, Uni ersity of Milano, Milano Italy

Received 22 April 2000; Accepted 25 May 2000; Revised 8 June 2000

Abstract. A method for assessing the risk for honeybees from pesticide exposure via pollen is proposed.Four pesticides, selected as markers, were monitored in pollen samples collected in two sampling areas,one located in an intensive agricultural area and the other far from direct pesticide impact. Analyticalresults were consistent with use patterns of the chemicals and their physico-chemical and persistenceproperties. For a preliminary estimate of bee exposure via pollen, both by ingestion and by contact, anexposure index was developed, based on physico-chemical properties, persistence and application rates.

Ž .On the basis of the exposure estimates and acute toxicological data ingestion and contact LD , Toxicity50Ž .Exposure Ratios TERs were calculated as indicators of the risk for honeybees due to this particular

Ž .exposure route. TER values were compared to Hazard Quotient HQ , calculated as the ratio betweenapplication rate and the LC value, according to European guidelines, showing a satisfactory agreement.50The advantage of the above described procedures is that the environmental fate of the chemicals, and notonly application rates, are taken into account. This approach may represent a preliminary tool for acomparative screening of the risk for pollinator insects due to this particular exposure route.

Keywords: honeybees; pesticides; risk assessment; integrated pest management; pollen

Introduction

The selection of environmentally friendly pesti-cides is a relevant issue for the management ofagricultural practices. In particular, it is very im-portant for high quality crops, such as those pro-duced in agreement with European Regulation

Ž .1257r1999 EC, 1999 , as well as for use in Inte-Ž .grated Pest Management IPM protocols.

For the assessment of the risk for non-targetorganisms, standard procedures have been devel-

* Corresponding author; E-mail: [email protected]

oped, such as those proposed by DirectiveŽ .91r414rEEC EEC, 1991 concerning the placing

of plant protection products on the marketŽEuropean and Mediterranean Plant Protection

.Organisation]EPPO, 1993 . These are generallybased on the concept of Toxicity Exposure RatiosŽ .TERs , i.e. the ratio between a toxicological endpoint and a Predicted Environmental Concentra-

Ž .tion PEC . Exposure is generally evaluated bymeans of predictive approaches, starting with sim-ple calculations based only on application rateand then, if necessary, refining these estimateswith more complex models based on usage pat-

Vighi et al.288

tern, physico-chemical properties and the envi-ronmental fate of a pesticide.

For the assessment of effects in terrestrialecotoxicology, honeybees are accepted in interna-tional procedures as representatives of the com-

Ž .plex group of pollinator insects EPPO, 1993 .For honeybees, the concept of Hazard QuotientŽ .HQ is used instead of TER. According to EPPO

Ž .guidelines EPPO, 1993 , a HQ is defined as theratio between the application rate of a pesticideŽ . Ž .AR, grha and the acute LD mgrbee . The50HQ utilises AR as a rough exposure indicator,due to the difficulty of calculating a PEC for beeexposure. Therefore, HQ doesn’t include anyevaluation of environmental fate.

Among the various routes of pesticide expo-sure for bees and other pollinator insects, pollenis particularly relevant, as it takes into accountthe possibility of exposing the whole bee colony.Nevertheless, little information is available on therole of pollen in pesticide exposure for bees and

Žother pollinator insects Russel et al., 1998;.Thompson and Hunt, 1999 .

In this paper, the exposure of honeybees topesticides via pollen is investigated and a proce-dure for risk assessment from this specific expo-sure route is proposed and applied to a series ofpesticides included in an IPM protocol.

Material and methods

The sampling areas

Pollen samples were collected in two differentareas of the Sondrio district in the northern part

Ž .of Italy from honeybee Apis mellifera L. hivesŽ .Fig. 1 . The first sampling station was locatednear Gualtieri village at 623m above sea level inVal Malenco and it is assumed to be free frompesticide impact because of the lack of any inten-sive agriculture. The second station is close toPonte in Valtellina at 500m above sea level. Inthis station, the sampling hives were located in-side a highly cultivated area with vineyards andan apple orchard of about 280 hectares. Thepollen was sampled by means of a pollen trapintroduced into the hives, one for each samplingstation, and collected weekly from March to theend of September.

Chemicals applied

During spring and summer fifteen different pes-ticides were applied in the cultivated areaŽ .Table 1 . Information on the application rate ofpesticides and of the approximate dates of the

Ž .treatments Table 2 were obtained from the co-operative ‘‘Ponte in Valtellina’’. Due to the or-ganisation of the co-operative, agricultural prac-tices are common and adopted, with a few differ-ences, by all farmers.

The main physico-chemical and toxicologicalproperties of the chemicals are shown in Table 1.

Ž .Air-water partition coefficients Kaw have beenŽcalculated from Henry’s law constant HsVPrS

3 .Pa m rmoles according to the following equa-tion:

Ž .KawsHrRT 1

Žwhere R is the gas constant Rs8.314 Pa3 .m rmoles 8K , and T is the absolute temperature

Ž .at 258C, Ts2988K .Ž .The octanol-air partition coefficient Koa is

an indicator of bioconcentration in terrestrialŽ .plant biomass Paterson et al., 1991 , therefore, it

can be taken as an indicator of pollen uptakecapability. Literature data on Koa are availablefor a very few chemicals, therefore, it has beencalculated from Kow and Kaw according to the

Ž .following equations Paterson et al., 1991 :

KowrKawsCorCw*CwrCasCorCasKoaŽ .2

where Co, Cw, and Ca are the saturation concen-Ž .trations vrv in octanol, water and air respec-

tively.

Chemical analysis of pollen samples

ŽFor analytical monitoring, four chemicals captan,.chlorpyrifos, methidathion and teflubenzuron

were selected among the fifteen active ingredi-ents utilised in the area. Main selection criteriawere:

v the amount utilised;v different physico-chemical properties, covering

a relatively wide range of environmental be-haviour;

Risk Assessment for Honeybees from Pesticide-Exposed Pollen 289

Ž .Figure 1. Schematic representation of the location of the two sampling sites 1 Gualtieri and 2 Ponte in Valtellina . For site 2,Ž .details on the cultivated surface shaded area and of the bee foraging area are shown. Foraging radius of 1 km and 2 km are

represented by dotted lines, the hive position is represented by a black point. Ten kilometres divide the two sampling station.

v the availability of suitable analytical methodsŽ .for the sampled matrix pollen .

A total of eight samples, collected in both areasafter each pesticide application period, were anal-ysed.

The samples were wrapped in aluminium foiland then stored at y208C until analysed. Threegrams of each sample were minced and ho-mogenised with NaSO , then extracted in a Soxh-4

Ž .let apparatus with n-hexane as solvent 4h ,

cleaned with 2 g of Florisil column using 10 ml ofŽ .hexane-ethyl acetate 90:10, v: v as eluent and 20

Ž .ml of hexane-ethyl acetate 70:30, v: v . Thisvolume was reduced by a rotary evaporator to 1ml for the GC-MS. Recoveries ranged from 70 to100%. The detection limits for the extracts were 1

Ž .ngrml corresponding to 0.33 mgrg of pollen formethidathion, chlorpyrifos and teflubenzuron,

Žwhile for captan it was 4 ngrml corresponding to.1.3 mgrg of pollen .

Vighi et al.290

Table 1. Physico-chemical and toxicological properties of the compounds utilised in the study area

DT50S VP soil LD LD50oral 50contact

MW grL Pa LogKow LogKaw logKoa days mgrbee mgrbee

Fungicidescaptan 300.6 5.1E-3 1.1E-5 2.5 I6.6 9.1 4 91 788dichlofluanid 333.2 1.3E-3 2.1E-5 3.7 y5.7 9.4 n.a. )100 )70dithianon 296.3 5E-4 6.6E-5 3.2 y4.8 8.0 n.a. )100 )100dodine 287.4 7.0E-1 -1.3E-4 1** y7.7 7.7 20 n.a. )11flusilazole 315.4 5.4E-2 3.9E-5 3.7 y7.0 10.7 n.a. )150 165hexaconazole 314.2 1.7E-2 1E-5 3.9 y7.1 11.0 122 )100 )100mancozeb ) 6E-3 -1E-4 2** -y9.4** )9.4** 10 )20 )16metiram ) 1.0E-4 -1E-4 0.3 -y9.4** )9.7** n.a. )40 )16myclobutanil 288.8 1.4E-1 2.1E-4 2.9 y3.8 6.7 66-282 )100 )362teflubenzuron 381.1 1.9E-5 8E-10 4.3 I8.2 12.5 84 n.a. )))))1000thiophanate-methyl 342.4 3.5E-3 9.5E-6 1.4 y6.4 7.8 10 n.a. )100

Insecticidesacephate 183.2 790 2.3E-4 1.12 y10.7 11.7 3 0.277 n.a.chlorpyrifos 350.6 7.3E-4 2.5E-3 5.3 I3.3 8.6 20 0.18 0.059methidathion 302.3 2.2E-1 4.49E-4 2.6 I6.6 9.2 7 0.21 0.13vamidothion 287.3 4000 1E-6** 1.08 y13.5 14.6 n.a. 0.15 0.56

For mancozeb and metiram, polymers with undefined molar weight, some properties are estimated values.Chemicals analysed in the experimental survey are reported in bold. In some cases, for soil DT values, figures50were selected as the most realistic in relation to climatic and environmental condition of the study area. Data from

Ž . ŽFinizio selection Finizio, 1998 , excepted for mancozeb, metiram, teflubenzuron which are from Tomlin Tomlin,. Ž .1997 *:MW. not defined; **: estimated values; n.a.: not available .

Eco-ethologic behaïour of honeybee population

In literature it is documented that honeybees canŽ .forage up to 6,000 meters Seeley, 1995 . Never-

theless this should be assumed as an extremescenario. Indeed if forage is enough abundant inthe short range, the foraging radius is usually less

Žthan 1]2 km. According to Pinzauti Pinzauti et.al., 1991 in an agroecosystem, where forage is

abundant, the radius does not exceed 1,000 me-ters.

Rivers, roads and railway are not real barriers.Nevertheless, due to the particular environmental

Žscenario railway, the heavy road traffic and the.Adda river as well as valley geomorphology , it is

reasonable to assume that honeybees forage onlyinside the orchard. Therefore the pollen collectedin the Ponte in Valtellina sampling station comes

Ž .from the treated area Fig. 1 and may be as-sumed as potentially contaminated.

Pollen examination showed that bees are for-aging both weeds and crop, when it was in flower.

It was assumed a comparable pesticide contami-nation for crop and weeds inside the orchard.

Results and discussion

Enïronmental concentration of pesticides:Analytical results

The results of chemical analysis are reported inTable 3. At the Gualtieri site, the four chemicalswere always below detection limits. At the Pontestation detectable concentrations of captan werefound in August and September. Traces ofteflubenzuron, close to detection limits and diffi-cult to quantify exactly, were found in the sampleat the end of May. These positive findings arein agreement with the treatment periods of the

Ž .two compounds Table 2 . The other two chemi-cals analysed were below detection limits in allsamples.

The interpretation of these results on the basisof predictable environmental behaviour is impos-


Table 2. Approximate dates of treatments, application ratesand total amount applied at each application in thestudy area

Application TotalDate of rate appliedtreatments Active compounds grha amount kg

25r3 mancozeb 2200 616methidathion 570 160

2r4 metiram 2150 60221r4 mancozeb 2200 616

myclobutanil 75 2127r4 metiram 2150 602

hexaconazole 36 107r5 dithianon 1125 315

vamidothion 670 18813r5 dodine 900 25219r5 mancozeb 2250 630

flusilazole 61 1725r5 mancozeb 2250 630

teflubenzuron 61 171r6 dodine 710 199

10r6 dithianon 1125 31516r6 mancozeb 2200 6161r7 dodine 900 252

acephate 1025 28714r7 thiophanate-methyl 750 2101r8 captan 1900 532

chlorpyrifos 665 18619r8 captan 1800 5045r9 dichlofluanid 1125 315

ŽThe total amount refers to the total cultivated area 280.hectares . The application rates for the same active ingredient

corresponding to different treatment dates could be slightlydifferent due to different commercial formulations used.Chemicals analysed in the experimental survey are reported inbold.

Table 3. Results of the chemical analyses in the twoŽsampling stations n.d.: all chemicals below

.detection limits

Ponte samples

Concentration GualtieriDate Chemicals mgrg samples

8r3r94 n.d. } n.d.6r4r94 n.d. } n.d.

27r5r94 teflubenzuron trace n.d.8r8r94 captan 4.5 n.d.

16r8r94 captan 4.0 n.d.25r8r94 captan 3.6 n.d.3r9r94 captan 6.6 n.d.

12r9r94 n.d. } n.d.

sible in quantitative terms. Nevertheless somequalitative comments can be made.

The major factors affecting the presence ofpesticides in pollen are the following:

v Given that the foraging area of bees lies mainlyin the treated area, the drift during or immedi-ately after treatment is the main contamina-tion route. Therefore, the amount of pesticideapplied and the treatment periods are the ma-jor factors affecting pesticide presence in theforaging area, while other factors indicating apossibility of air transport over a relatively long

Ž .range e.g. volatility are less relevant;v A second important factor is the capability of a

chemical to be incorporated into pollen. Thisproperty may be estimated from the logKoa,which is related to the bioconcentration capa-

Žbility in the vegetal biomass Paterson et al.,.1991 ;

v Finally, a third significant parameter is persis-tence. The only available persistence data arethe soil DT values which cannot be related to50persistence in pollen, nevertheless they couldbe taken not as a real quantitative figure butas a rough qualitative and comparative indica-tor of the intrinsic resistance to degradation.Anyway, persistence can be assumed as rele-vant to explain disappearance in the medium-long term, but not as an indication of contami-nation potential immediately after application.

In order to combine application rate and persis-tence, a time weighted average was calculatedtaking into account that the residual amount aftera given time ‘‘t ’’ is related to persistence as fol-lows:

AM sAM eyktt i

where:AM sresidual amount at time ttAM sinitial amounti

ksln 2rDT50

Ž .A time weighted average TWA can be calcu-lated by integrating from 0 to t the previousequation:

Ž yk t .TWAsAM 1ye rkti

Vighi et al.292

For repeated applications, TWA can be calcu-lated as follows:

yk tŽ .TWAs AM 1yei1

yk Ž tyt1.Ž .qAM 1ye rkti2

were AM and AM are the amounts applied ati1 i2the different applications and t is the time be-1tween the two applications.

TWA calculated for the active ingredients atdifferent time intervals after first application areshown in Table 4.

Captan was analysed in samples collected from7 up to 33 days after the first application, whereTWA range from 1100 up to more than 600 grha.It is characterised by a medium logKoa valueŽ .9.1 and was the only chemical found in de-tectable amounts in pollen.

In the same period, TWA for chlorpyrifos areŽsignificantly lower from 600 up to about 400

. Ž .grha . This, together with the lower logKoa 8.4could justify the absence of positive findings.

TWA of teflubenzuron at the sampling timewas 61 grha. In spite of the low amount, it wasdetected, even if in small traces. It must be high-

Ž .lighted that Koa of teflubenzuron 12.5 is threeorders of magnitude higher in comparison with

the other analysed chemicals. This finding seemsto support the role of logKoa in affecting pesti-cide assumption by pollen.

Finally, methidathion was sampled 12 days af-ter the treatment, with a TWA of about 300

Ž .grha. The log Koa value 9.2 is comparable tothose of captan, thus, the lower amount couldjustify the lack of positive findings.

Deëlopment of an exposure index

A qualitative indicator of the probability of expo-sure for honeybees and other pollinator insectsmay be proposed through an exposure index based

Žon the main exposure factors amount applied,.logKoa and persistence .Ž .An exposure index EI can be developed by

giving a score to TWA and to log Koa, classifiedŽ .into discrete classes Table 5 .

The exposure index can be calculated as fol-lows:

EIsS )STWA LogKoa

where S and S are the scores attributedTWA LogKoato TWA and LogKoa respectively.

The result is a discrete classification, with 24possible final scores ranging from 0.01 and 1024.

Ž .Table 4. Time weighted averages TWA of chemical amounts used in the study area

Days after treatment

Chemicals 2 7 14 21 28 35 120

Fungicidescaptan 1607 1100 714 711 693 629 179diclofluanide 811]1112 21]1082 29]1039 154]1000 114]961 93]925 27]607dithianon 811]1112 21]1082 29]1039 154]1000 114]961 139]1232 54]1154dodine 868 800 714 639 575 646 568flusilazole 12]17 6.4]16 3.5]16 2.3]15 1.8]15 1.4]14 1]32hexaconazole 36 36 36 32 32 32 26mancozeb 2054 1743 1407 1157 1118 1246 1314metiram 1550]2125 07]2064 39]1986 296]1911 364]2064 350]2350 104]2196myclobutanil 75]75 71]75 71]75 68]71 64]71 64]71 43]65teflubenzuron 61 61 57 57 54 54 38thiophanate-methyl 700 593 479 396 332 282 90

Insecticidesacephate 821 507 304 211 157 125 37chlorpyrifos 639 589 525 471 425 382 157methidathion 518 411 311 239 193 161 48vamidothion 486 254 136 89 68 54 16

Data are expressed as grha of active ingredient at different time intervals, including repeated applications. If DT values50were not available a default range of 2 to 60 days was assumed.


Table 5. Scores assigned to TWA and LogKoa for theŽ .calculation of the Exposure Index EI

TWA LogKoa

grha score score

-10 0.1 -5 0.110]20 0.2 5]6 0.220]50 0.5 6]7 0.550]100 1 7]8 1

100]200 2 8]9 2200]400 4 9]10 4400]800 8 10]11 8800]1500 16 11]12 16)1500 32 )12 32

The approach is largely arbitrary, nevertheless, itcould be utilised for a rough preliminary classifi-cation of the probability of pollen contamination:

Class A: chemicals with very high probability ofpollen contamination in large amount, with

Ž .high 7 days TWA )800 grha and high logŽ .Koa )11 ; total score is G256;

Class B: chemicals with high probability of pollencontamination, with total score G64;

Class C: chemicals with significant probability ofpollen contamination, at least in trace, withtotal score G8;

Class D: chemicals with low probability of de-tectable pollen contamination, with total scoreG0.4;

Class E: chemicals with negligible probability ofdetectable pollen contamination, with totalscore -0.4.

Captan, detected at the concentration of a fewmgrg, behaves to Class B, while teflubenzuron,

Ž .detected in traces below 1 mgrg behave toClass C.

In order to produce quantitative data, to beŽ .compared with toxicological end points LD ,50Ž .for calculating a toxicologyrexposure ratio TER ,

an approximated preliminary PEC could be at-tributed to each class as follows:

Class A: PECs100 mgrgClass B: PECs10 mgrgClass C: PECs1 mgrgClass D: PECs0.1 mgrgClass E: PECs0.01 mgrg

These figures must not be taken as real PECs,but only as reference values, valid in comparativeterms for a comparative screening of pesticiderisk for pollinator insects.

The index may be calculated for the shortterm.

The fifteen chemicals utilised were classifiedaccording to this approach as reported in Table 6.Classification is referred to choosing 7 days TWA,assumed as exposure in the short term, after firstapplication, and to 120 days TWA, assumed aslong term exposure, taking into account repeatedapplications.

Risk assessment

A first step to assess the risk for bees is theevaluation of bee exposure through pollen, start-ing from the estimated figures of PEC of pesti-cides in pollen.

To calculate bee exposure, the following as-Žsumption can be made Contessi, 1983; Seeley,

.1985 :

}the amount of pollen imported in a hive isabout 40 kg per year and the estimated beepopulation is 250,000 individuals per year; thusthe amount of pollen per bee is about 160 mg;

}taking into account that a larva eats about 130mg of pollen in about 6]8 days of develop-ment, and that the weight of a larva rangesfrom less than 1 mg up to about 100 mg at theend of development, assuming as constant the

Žfood assumption as percentage of the body.weight during the development, it could be

assessed that the larva eats about 70% of itsbody weight per day; while adults eat about 30mg in about 5]6 days. As a consequence, a 100mg larvae eats about 70 mg of pollen per dayand an adult about 5 mg per day. Thus, theestimated amount of xenobiotics ingested in aday is 70 mg)PECr100 mg body weight forlarvae and 5 mg)PECrbee for adults.

}the amount of pollen collected by a bee isabout 300 mgrday, thus the amount of xenobi-otics collected in a day is 300 mg) PEC. Thiscan be assumed as contact exposure and onlyworker bees are exposed.

The data reported are indicative mean valuesand represent a necessary simplification of the

Vighi et al.294

Ž .Table 6. Classification of the fifteen utilised chemicals according to the proposed exposure indexes short and long term

Scores EI Class

TWA TWA Short Long Short LongŽ . Ž .7d 120d LogKoa term term term term

Fungicidescaptan 16 2 4 64 8 B C

Ž .diclofluanide T 2d 8 0.5 4 32 2 C D1r2Ž .diclofluanide T 16 8 4 64 32 B C1r2

Ž .dithianon T 2d 8 1 2 16 2 C D1r2Ž .dithianon T 60d 16 16 2 32 32 C C1r2

dodine 16 8 1 16 8 C CŽ .flusilazole T 2d 0.5 0.1 8 4 0.8 D D1r2Ž .flusilazole T 60d 1 0.5 8 8 4 C D1r2

hexaconazole 0.5 0.5 16 8 8 C Cmancozeb 32 16 4 128 64 B B

Ž .metiram T 2d 16 2 4 64 8 B C1r2Ž .metiram T 60d 32 32 4 128 128 B B1r2

Ž .myclobutanil T 2d 1 0.5 0.5 0.5 0.25 D E1r2Žmyclobutanil T 1 1 0.5 0.5 0.5 D D1r2

teflubenzuron 1 0.5 32 32 16 C Cthiophanate-methyl 8 1 1 8 1 C D

Insecticidesacephate 8 0.5 16 128 8 B Cchlorpyrifos 8 2 2 16 4 C Dmethidathion 8 1 4 32 4 C Dvamidothion 4 0.2 32 128 6.4 B D

For some chemicals, due to the lack of persistence data, two figures are reported corresponding to the minimum andmaximum persistence score.

complex situation of the bee population. Never-theless they can be assumed as reliable figures fora ‘‘worst case’’ scenario. With these data theestimated quantities of pesticides ingested byadult bees and larvae or collected by worker beesare shown in Table 7.

A preliminary risk assessment for honeybeesthrough pollen can be made by calculating theratio between toxicity data and pollen exposureŽ .TER: Toxicity Exposure Ratio . Only short term

TERs have been calculated because only acuteŽ .LD contact and oral are available. According50

Ž .to EPPO 1993 , as the main route of hazardousexposure to acutely toxic compounds is throughcontact action, the contact LD should be most50important for insecticides, while the oral LD50should be more relevant for the assessment ofcompounds not acutely toxic, such as fungicides.In the TER calculation, both contact DT and50oral DT were used if available.50

Table 7. Calculated exposures through pollen via food and contact for the four exposure classes

Amount of pesticide ingested with Amount of pesticide pickedfood in a day up with pollen in a dayPollen

PEC Larvae of 100 mgŽ .mgrg Adults body weight Worker bees

Class A 100 0.5mgrd 7mgrd 30mgrdClass B 10 0.05mgrd 0.7mgrd 3mgrdClass C 1 0.005mgrd 0.07mgrd 0.3mgrdClass D 0.1 0.0005mgrd 0.007mgrd 0.03mgrdClass E 0.01 0.00005mgrd 0.0007mgrd 0.003mgrd


Toxicity data are available only for adults bees.ŽIt is likely that some chemicals in particular

.insect growth regulators could be more toxic tolarvae. In these cases, data from bee brood feed-

Ž .ing tests Wittman and Engels, 1981 should beused. Nevertheless, at present, there is too little

Ž .data available EPPO, 1993 , therefore, a TERhas been calculated for larvae too, using the sameoral LD in order to obtain at least an indicative50and comparative value.

According to the toxicity data of Table 1, theproposed TERs are reported in Table 7. In rela-tion to the higher exposure level, the risk forlarvae is one order of magnitude higher in com-parison to adults. The assessment could be re-fined if toxicity data on larvae were available.

For the chemicals examined, a risk of acuteŽ .toxic effects TERF1 from pollen exposure has

Žbeen calculated for vamidothion larvae and adult. Ž .contact , chlorpyrifos adult contact and acephate

Ž . Žlarvae . Values close to a risk threshold 1-.TER-10 have been calculated for highly toxic

organophosphorous insecticides and values ofsome concern has been evaluated for fungicides

Žwith very high application rates mancozeb and.metiram . All other chemicals shows a TER higher

than 100, indicating a low or negligible risk.

Calculated TERs have been compared with theŽ .traditional hazard quotient HQ . The results of

Ž .HQ oral and contact are reported in Table 8.Currently applied triggers for HQ are the fol-

lowing:

HQ-50: harmless for bees;50-HQ-2500: slight to moderately toxic for bees;

HQ)2500: dangerous for bees.

As it could be expected, the comparison betweenthe two approaches shows a relatively good agree-ment. Chemicals with high HQ, such as organo-phosphate insecticides, are also characterised bylow TERs, while low HQs correspond to very highTERs. Nevertheless, some differences in the clas-sification are evident. In particular, chemicals with

Žhigh logKoa see, for example, vamidothion or.teflubenzuron are classified as more dangerous

by TERs in comparison to HQ. This confirmsthat, for the particular route of exposure consid-ered here, i.e. via pollen, the proposed methodallows risk to be assessed on the basis of exposureindicators more related to features relevant forenvironmental distribution and fate than simpleapplication rates.

Table 8. TERs and hazard quotients for the fifteen compounds utilised in the Ponte in Valtellina orchards

TERs for TERs foringestion adults ingestion larvaeŽ . Ž .LD rPEC LD rPEC TERs for contact HQ HQ50oral 50oral

Ž .short term short term LD rPEC oral contact50contact

Fungicidescaptan 1820 130 262 20 2.4dichlofluanid )2E4])2E3 )1400])140 )233 -11 -16dithianon )2E4 )1400 )33 -11 -11dodine } } )3.7 } -82flusilazole )3E5])3E4 )21400])2140 5500]550 -0.4 0.37hexaconazole )2E4 )1400 )330 -0.4 -0.4mancozeb )400 )28 )5 -110 -137metiram )800 )57 )5 -54 -134myclobutanil )2E5 )14000 )12000 -0.8 -0.2teflubenzuron } } )3300 } -0.06thiophanate-methyl } } )330 } -7.5

Insecticidesacephate 5.5 0.4 } 854 }

chlorpyrifos 36 2.5 0.19 3694 11271methidathion 42 3 0.4 2714 4392vamidothion 3 0.2 0.19 4466 1198

Vighi et al.296

Conclusions

The described approach must be taken as a provi-sional methodological suggestion. Many quantita-tive assumptions for the development of the expo-sure index are largely arbitrary and are based ona few experimental data. More extensive experi-mental monitoring would result in a better cali-bration. At present, data on pollen contamination

Žare very scarce in the literature Thompson and.Hunt, 1999 and the few data available are never

related to pesticide use in the examined area,thus, they are not applicable to the describedprocedure.

A further controversial item is the meaning ofKoa values. The role of Koa for estimating bio-concentration potential in terrestrial plantbiomass is widely accepted. Even if it does nottake into account specific mechanisms, such astranslocation in different part of the plant, it iscurrently assumed as an indicator of bioconcen-tration in plant tissues, as Kow, with comparableconceptual limitations, is assumed as an indicatorof bioconcentration in animal tissues. It must alsobe taken into account that pollen has a high lipidcontent, thus Koa could be a significant indicatorfor presence in this particular plant compartment.

Nevertheless, some aspects need to be betterclarified with larger experimental evidence.

.1 The calculation of Koa as a ratio between Kowand Kaw is mathematically correct, but hasbeen verified experimentally on few chemicalsand should be confirmed on a wider range ofmolecular properties. E.g., for very solublechemicals, high Koa values can be calculatedeven with low Kow and medium vapour pres-

Ž .sure see, for example, vamidothion . Thus,there is the need for more experimental dataon Koa.

.2 As a consequence of the first point, the rela-tionship between Koa and bioconcentration inplants has been verified, on sound experimen-

Žtal bases, with relatively few chemicals Pater-.son et al., 1991 . Consequently, the classes of

bioaccumulation potential used for the devel-Ž .opment of the index Table 4 are largely

arbitrary.

The calculated TERs are in agreement with theHQ, with some outliers. Anyway, the describedapproach does not represent an alternative toHQ. The HQ is applicable for general prelimi-nary comparative assessment of pesticides, with-out any connection with specific environmental oragronomic conditions. Therefore, it is suitable fora preliminary assessment such as those requiredby regulations for the registration of new com-

Žpounds see for example European Directive.91r414rEEC . It must be remembered that, even

if HQ is currently applied for regulatory pur-poses, real experimental validations of this ap-proach are still not available.

The main advantages of the approach de-scribed in this paper are:

v it can be applied to specific scenarios, where adefined amount of pesticides is applied or issupposed to be applied on a given area; there-fore, it could be useful for selecting moreenvironmental friendly pesticides in specificenvironmental and agronomic conditions, inparticular if higher environmental care is re-quired, such as in Integrated Pest Manage-ment protocols;

v even if pollen is not the only exposure route, itis particularly relevant given that, throughpollen, all developmental stages of the beecolony are exposed; the approach allow to cal-culate larval exposure, thus, a more significantrisk assessment for bee populations can becalculated; the assessment could be improvedif toxicity data on larvae would become avail-able;

v even if estimated PEC cannot be assumed asrealistic concentrations, exposure is not onlybased on application rates; other fate parame-

Ž .ters persistence, bioconcentration potentialare taken into account; moreover, TWA can becalculated at different time intervals in orderto assess potential long term exposure of thebee colony, for persistent or repeatedly appliedpesticides.

Acknowledgement

The authors wish to thank Dr. RobertaMaffescioni for her help in collecting samples.


References

Ž .Contessi, A. 1983 . Le api: biologia, alleämento, prodotti.Bologna, Edagricole.Ž . Ž .EC 1999 . Reglement CE n8 1257r1999 du Conseil du 17`

mai 1999 concernant le soutien au developpement rural´par le Fonds europeen d’orientation et de garantie agri-´

Ž .cole FEOGA et modifiant et abrogeant certains regle-`ments. G.U. n. L 160r80.

Ž . ŽEEC 1991 . Directive 91r414 CEE of 15 July 1991 G.U..CEE n.8 L230 19 August 1991 .

European and Mediterranean Plant Protection Organisation.Ž .1993 . Decision-making scheme for the enïronmental riskassessment of plant protection products. Bulletin

Ž .OEPPrEPPOr 23 , 1]157.Ž .Finizio, A. 1998 . Definizione di criteri di classificazione del

rischio ambientale di prodotti fitosanitari, con particolareŽ .riferimento agli effetti su organismi non bersaglio parte II .

Rapporto ANPA N.ro 97-58-RC050-75.Ž .Paterson, S., Mackay, D., Bacci, E. and Calamari, D. 1991 .

Correlation of the equilibrium and kinetics of leaf-airexchange of hydrophobic organic chemicals. Enïron. Sci.

Ž .Technol. 25 5 , 866]71.

Pinzauti, M., Frediani, D., Biondi, C., Belli, R., Panizzi, L.,Ž .Cosimi, C. and Zummo, V. 1991 . Uses of honeybees for

Ž .monitoring environmental pollution in Italian . Analysis8, 355]405.

Ž .Russel, D., Meyer, R. and Bukowski, J. 1998 . Potentialimpact of microencapsulated pesticides on New Jersey

Ž .apiaries. American Bee J. 138 3 , 207]10.Ž .Seeley, T.D. 1985 . Food collection. In J.R. Krebs and T.

Ž .Clutton-Block eds Honeybee Ecology. A Study of Adapta-tion in Social Life, pp. 80]9. Princeton, NJ: PrincetonUniversity Press.

Ž .Seeley, T.D. 1995 . The Wisdom of the Hi e: The SocialPhysiology of Honeybee Colonies. Harvard University Press.

Ž .Thompson, H.M. and Hunt, L.V. 1999 . Extrapolating fromhoneybees to bumblebees in pesticides risk assessment.

Ž .Ecotoxicology 8 3 , 147]66.Ž .Tomlin, C.D.S. 1997 . The Pesticide Manual: A World Com-

Ž a . Ž .pendium. 7 ed. British Crop Protection Council edsFarnham, UK.

Ž .Wittman, D. and Engels, W. 1981 . Development of testprocedure for insecticide-induced brood damage in honey

Ž .bees in German . Mitteilungen der Deutschen Gellschaft furAllgemine und Angewandte Entomologie 3, 187]90.

Documents

Risk Assessment for Honeybees from Pesticide-Exposed Pollen