Environmental Toxicology and Chemistry, Vol. 16, No. 9, pp.

19771982, 1997 1997 SETAC Printed in the USA .00 0730-7268/97 $6.00

Hazard/Risk Assessment
A UNIFORM PROCEDURE TO ESTIMATE THE PREDICTED ENVIRONMENTAL CONCENTRATION OF THE RESIDUES OF VETERINARY MEDICINES IN SOIL
KATRIN R.I. SPAEPEN, LEO J.J. VAN LEEMPUT,* PETER G. WISLOCKI and CHRISTIAN VERSCHUEREN
European Federation of Animal Health, Brussels, Belgium Janssen Pharmaceutica NV, B-2340-Beerse, Belgium Merck Research Laboratories, Rahway, New Jersey, USA (Received 2 July 1996; Accepted 21 January 1997) AbstractNew guidelines to assess the environmental risk of veterinary medicines (VMs) have been proposed in the European Union. As in any risk assessment, exposure is a factor of critical importance. The predicted environmental concentration (PEC) in soil therefore is a pivotal decision parameter in the assessment procedures for VMs. As presently proposed, phases I and II of the assessment require a PEC at an appropriate level of renement. Hence, it is necessary to calculate the PEC of VMs in a uniform way. The PEC for VMs depends on animal husbandry practices, on the use pattern of the product, on its kinetics in the treated animals and in soil, on physiological characteristics of these animals, and on the codes of good agricultural practices of excreta collecting, storage, and of fertilizing land. In this paper, basic calculations are presented to estimate the PEC from the concentration of residues in manure, the quantities of manure spread per unit land area, and the depth of incorporation into soil. Expanded calculations allow renements for treatment of only part of the group of animals, metabolism, periods in house, transformation in manure or soil, and repeated application. Examples presented illustrate the dependence of the PEC on various parameters. The examples allow the examination of the regulatory framework of the phase I PEC and an alternative approach to judge the persistence during manure storage or the persistence in soil is proposed. KeywordsVeterinary medicines Predicted environmental concentration Soil Manure Environmental risk

INTRODUCTION

People keep animals as companions, to assist them in work and as a source of food. Healthy animals are the most suited for all these purposes. Veterinary medicines (VMs) help to protect the health and to ensure the well-being of our animals. VMs are licensed for use by regulatory authorities if they comply with the scientic criteria on quality, efcacy, and safety. With regard to the safety criterion, the authorities consider safety to the treated animal, the consumer, and individuals handling the product during the course of treatment. In addition to safety for the treated animals, for the users and the consumers, recently the safety of VMs for the environment has become a matter of increasing public scrutiny and legal requirements. The legal requirements in the European Union (EU) on the environmental risk assessment of VMs are contained in Commission Directive 92/18/EEC [1]. Specic provisions require manufacturers to demonstrate the absence of any negative impact on the environment from residues of VMs. This should be done in two phases. In the rst phase (phase I), an expert has to assess the potential exposure to the environment, taking into account all relevant physicochemical parameters of the active substance, its metabolism, the target species, and the pattern of use of the derived product. This should include an estimate of the predicted environmental concentration or PEC. Should the PEC exceed a certain value, for example 10 g/kg soil as presently imposed by the regulatory authorities in the EU, further fate and effects tests must be carried out to allow an evaluation in a phase II. Detailed guidelines to assist applicants in the conduct of investigations and the assessors in
* To whom correspondence may be addressed. 1977

their evaluations are currently being elaborated for the two phases of the environmental risk assessment [2]. The elaboration of the PEC is an important element in phase I and phase II. The objective of the present document is to contribute to establishing uniform methods to demonstrate the safety of VMs for the environment, by proposing a single method for the estimation of the PEC in soil. Such a method should provide continuity across international borders. The method is logical and conservative but does not intend a renement or a validation beyond the level needed for an environmental risk assessment of VMs in the present regulatory framework. Residues of VMs can enter the environment with the excreta of treated animals when manure is spread on agricultural land to fertilize it. Soil, thus, is the environmental compartment primarily exposed. Based on rates of incorporation of manure into soil and the knowledge of the levels of drug residues in manure, the amount of drug residues in soil can be calculated. In the EU, good agricultural practice regulations insure that land is not overfertilized with respect to the nitrogen or phosphorus content of manure used as fertilizer. These nitrogen and phosphorus limits thus directly determine the maximum allowed rates of manure incorporation. In the approach presented below, the existence of these limits will be exploited to estimate the worst case concentrations of residues of VMs. Practices of excreta collection and storage of manure, their spreading onto land, and incorporation into soil display wide regional variations. These parameters, however, strongly inuence the PEC. To prevent PEC calculations from being done in many different ways and to different degrees of detail, a uniform PEC calculation system, based on existing scientic literature and on expertise from academics and industry is

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Environ. Toxicol. Chem. 16, 1997

K.R.I. Spaepen et al. Table 1. Some relevant characteristics of the various types of animals included in the PEC calculation routine and the linked database

presented. This work was undertaken under the auspices of the European Federation of Animal Health (FEDESA) for this purpose. The broad use of a uniform PEC calculation scheme will assist the harmonization of environmental assessments and will generate comparative data for many products, this, in turn, enabling validation and adaptations where necessary. The proposed scheme provides a sequence of standard equations and a database currently containing information on three major agricultural species, cattle, pigs, and poultry. The database also contains information on agricultural practices and relevant regulations for various regions in the EU. This information is presented in a summarized form in this paper. The equations and the database are also linked in a computer program, which will be commercially available. If necessary, the database can be expanded to include other member states, other animal species, and other agricultural practices and can be adapted if necessary to changing regulatory requirements.
CALCULATIONS

Animal type Slaughter calf Dairy cow Beef bullock Bull Piglet Fattening pig Adult pig Sow and litters Broiler chicken Laying hen References

N BW (year 1) (kg)
4 1 1 1 6 2.5 1 1 9 1 [6]

PE (kg/place/ year)

PN (kg N/ place/ year)

10.9 77.29 52.64 77.86 3.35 9.59 17.12 24.96 0.21 0.56 [811]

PP (kg P2O5/ place/year)

5.28 39.08 16.24 29.12 2.5 8.32 12.45 16.38 0.38 0.58 [8, 1013]

4,660 160 20,391 500 9,185 500 20,075 1,200 754 20 1,764 95 2,829 130 4,928 100 37.2 1.3 67.5 2 [6] [7,8]

Two sets of calculations are presented: basic and modied calculations. For the basic sequence of calculations, the following are assumed: (1) The active substance in the administered VM is completely excreted in urine and feces. At this stage, transformation by metabolism is not taken into account. Thus a total residue concept is adopted, as employed in the safety assessment of residues of VMs in food. (2) The treated animals are housed. (3) The excreta of all animals are collected and stored combined. (4) Manure is spread on land once a year. The modied equations, to be presented further on, allow one to approximate real practice more closely where possible and appropriate. The calculation routine proceeds through a number of steps.

Basic calculations
In step 1 the total quantity of active ingredient (Q, mg per year per place) given to the animals on each place available in an animal housing location is calculated from the individual dose rate (ID, mg per kg body weight), the body weight of the target animal type (BW, in kg), the number of individual treatments per animal (T, per animal), and the number of animals raised per year on each place in the animal housing location (N, per year per place)

without correction, which is justied because the water content of combined excreta in most cases exceeds 90%. In step 3 the maximum quantities of manure applied to land are calculated. A direct way to do so could have been to collect data on manuring rates from agricultural practice. Instead, an indirect but more conservative approach was preferred. In many regions of the EU, regulations exist on maximum inputs of fertilizing substances, in an attempt to reduce pollution due to excessive release of nutrients. These regulations state the maximum amount of nitrogen (AN, kg N per hectare per year) and/or phosphorus (AP, kg P2O5 per hectare per year) that can be applied. These limits can be transformed into maximum manuring rates if the yearly production of nitrogen (PN, kg N per place per year) and phosphorus (PP, kg P2O5 per place per year) and the yearly manure output (PE) are known for the animal species of interest. The limit rates for nitrogen (MN, kg excreta per hectare per yearnitrogen) and phosphorus (MP, kg excreta per hectare per yearphosphorus) are

MN MP

AN PN AP PP

PE PE

(3) (4)

ID

BW

(1)

ID and T are specic for the product and the target animal and must be available as an input for the calculations. For BW, typical values per animal type are used. N depends on husbandry. For example, broiler growout takes about 6 weeks. So, on each place in a broiler house, about nine birds can be raised in a year. Thus, N will be 9 per year and per place, representing the turnover rate on the farm. Information was obtained from literature and through consultation with relevant experts. The information was included in the database and is shown in Table 1 in summarized form. In step 2 the concentration of residues of the active ingredient in the combined excreta (CE, mg per kg excretafeces and urine together) is calculated from Q and the yearly output of excreta (PE, kg per place per year) CE Q /P E
(2)

The allowed quantities depend on the agricultural use of the land. The required information for the variables in Equations 3 and 4 were obtained as stated above (Tables 1 and 2) and loaded in the database linked to the calculation routine. Either the limit for nitrogen or the one for phosphorus is the most restrictive and should therefore be used. The calculation routine in the computer program selects the most restrictive rate, M (kg excreta per hectare per year), to continue. In step 4, step 2 and step 3 are combined to obtain the amount of active substance-related residues applied per unit area of land (CSA, mg per hectare per year)

CSA

CE

(5)

Values for PE were obtained as stated above. Averages are shown in Table 1 and included in the linked database. Where the source contained output data in liters, the value was entered

Note that on combining Equation 2 and Equation 3, the variable PE in fact cancels. CSA can therefore be directly obtained by combining Q and the most relevant ratio from AN/PN and AP/PP (in places per hectare). The procedure involving PE is preferred, however, for clarity and possible expansion of the calculation routine. In step 5 CSA is transformed into concentrations in the

The PEC of residues of veterinary medicines in soil Table 2. Data on the maximum allowed amount of nitrogen (AN) and phosphorus (AF) that can be applied to land per year for soil fertilization in various member states of the European Union

Environ. Toxicol. Chem. 16, 1997

1979

Expanded calculations
Some modications of the basic equations can be used to rene the PEC in soil. Modication 1 is applied if only a fraction (FT, fraction of the animals receiving treatment) of the animals delivering excreta to the manure collection system is treated. Equation 2 then modies to

Location Belgium Denmarka France Germany Italyf Spaing The Netherlands UK

Land type Arable Grass Maize Arable Grass Maize Arable/maize Grass Arable/grass/maizec Arable/grass/maized Arable/grass/maizee Arable/grass/maize Arable Grass Maize Arable/maize Grass Arable Grass Maize Arable/grass/maize

AP AN (kgP2O5/ (kg ha/ N/ha/ year) year) Reference

100 100 120 27 10 35 b 210 120 700 70 100 100 110 150 230 80 50 170 250 170 139 125 140 200 350 240 160 200 600 100 140 200 250 [14] [15] [16] [17] [17] [17]

CE

Q PE

FT

(9)

Modication 2 allows inclusion of experimental evidence on the material balance of the substance in the animal. If the parent substance, a metabolite or a transformation product in general accounts for a fraction FM (fraction of the total administered substance metabolized) of the administered dose, Equation 2 modies to

CE
[18] [19]

Q PE

FM

(10)

Europeh
a b

170

[20]

Average values depending on crop type. No value specied. c North Rhine Westfalia. d Bremen, Schleswich-Holstein. e Nieder Sachsen. f Personal communication from Dr. Salvo, Assoconimi, with the Ministry of Agriculture in Rome, Italy. g Personal communication from Dr. Aragon, Veterindustria, with the Ministry of Agriculture in Madrid, Spain. h Value used for the calculation of the PEC in the EU.

This modication must be considered for every major fraction, that is, all those exceeding, for instance, 20% of the administered dose. In modication 3 Q from Equation 1 can be replaced by a reasonable alternative estimate Q* (mg per year per place) of the yearly dose per animal ending up in the excreta collection system. This modication can, for instance, be considered in a situation where the animals are housed for part of the year only. Q* then will encompass all treatments given in house. Further, the part of the housed period in a year (FH, fraction of a year when animals are housed) must be estimated. Then, Equation 2 modies to

CE

Q* PE FH

(11)

ploughed soil (CSV, mg per kg soil and per year). To do this, the weight of the ploughed layer (W, kg per hectare) is calculated from the soil bulk density ( , kg per m3) and the volume of the ploughed layer (V, m3 per hectare). The latter depends on the depth of the furrow (D, in cm). The relation between V and D, after converting cm into m and hectare into m2 is

D 100

100

100

100

(6)

The weight of the ploughed layer W is

(7)

D depends on regional agricultural practice. For instance, a furrow depth of 25 cm can be used when the land is ploughed [3]. In the absence of soil disturbance, it can be assumed that the substance distributes over the top 5 cm of the soil [2]. Relevant information on ploughing depth was obtained as stated above and loaded in the linked database. The soil bulk density obviously changes with soil texture, but an arbitrary average value of 1,500 kg/m3 was used [2]. The PEC in soil is CSV CSA W M
(8)

An approach with a Q* also can be adopted when data on disease incidence or alternative use patterns can be documented. Use of extension 1 allows accounting for degradation of the substance in stored manure, if data on transformation kinetics, such as a 50% disappearance time (DT50E, in days), are available. First-order degradation is assumed for the calculation, setting the half-life (t1/2) equal to DT50E. In practice, transformation kinetics are not necessarily rst order but might follow more complex mathematical forms. Further, within a given transformation model, the rate parameters are likely to depend on variables such as temperature, nutrient status, and redox conditions. Within the concept of the present model, a restriction to rst-order kinetics and a neglect of the variability of the rate parameters is justied, the more because the model should be applicable at a stage in the regulatory process where only a limited set of data exists. When more detailed information on the transformation kinetics is available, the equations below can be easily adapted. The time available for degradation (tE, residence time in excreta in days) is that between animal treatment and manure spreading. The concentration in manure at spreading in this case is

CE

Q PE

e(

ln2/DT50E ) t E

(12)

Equation 8 accounts for the weight of the applied manure (M) itself to derive CSV.

Use of extension 2 allows calculation of the concentration in soil at a given residence time after incorporation (ts, residence time in soil in days), provided experimental evidence is available on the degradation of the substance in soil, such

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Environ. Toxicol. Chem. 16, 1997

K.R.I. Spaepen et al. Table 3. Results of PEC calculations for some animal types and treatment schemes involving the administration of daily doses of 1 or 5 mg per kg body weight (BW) for 1 or more (T) d PEC ( g/kg) Animal type

as a 50% disappearance time (DT50S, in days). Application of the same mathematics as used to arrive at Equation 12, taking into account the remarks on the rst-order approach and the disregard of rate parameters as explained above for manure and applicable to transformation in soil too, gives

T
1 1 1 7 40 1 10 60

1 mg/kg BW 2.62 0.29 2.51 17.54 100.22 1.61 16.07 96.45

5 mg/kg BW 13.06 1.45 12.53 87.70 501.2 8.04 80.37 487.24

CSV [tS ]

CSV [t0 ]

e(

ln2/DT50S ) t S

(13)
Slaughter calf Dairy cow Broiler chicken Piglet

Because the same kinetic model was introduced for the degradation in manure and in soil, the attenuating effect on the concentration of the residues will be identical in both matrices and can be described by a factor FD (attenuation factor caused by degradation in manure or soil)

FD

e(

ln2/DT50 ) t

(14)

In some instances, it can be appropriate to calculate a mean concentration in soil (CSV, mg per kg soil) over a certain period, for instance to evaluate the exposure over the duration (tT, residence time in an ecotoxicity experiment in days) of a terrestrial ecotoxicity test. In extension 3, this can be done with the equation
CSV [tT ]

The manuring was restricted to 170 kg N per year and per hectare. The ploughing depth was 25 cm.

CSV [t0 ]

DT50S [1 e( ln 2 tT

ln2/DT50S ) t T

(15)

Finally, extension 4 allows estimation of the concentration in soil on a piece of land where residues of the same substance are applied repeatedly with a certain interval (i, time interval between repeated applications in days). Basic pharmacokinetic theory [4] teaches that in such cases a steady-state condition will be reached after a number of applications. The concentrations then uctuate between a calculable maximum and minimum. This is the case for all substances with a nite DT50. These concentrations are obtained as follows:

CSV [SS, max]

and

CSV

1 1

e(

ln2/DT50S ) i

(16)

CSV [SS, min]

CSV

1 1

e(

ln2/DT50S ) i

e(

ln2/DT50S ) i

(17)

RESULTS AND DISCUSSION

The performance of the PEC calculation routine is illustrated and discussed for an imaginary substance, assuming the following parameters: treatments of slaughter calves (160 kg), dairy cows (500 kg), piglets (20 kg), or broilers (1.3 kg); individual dosage rates (ID) of 1, 5, or 10 mg/kg body weight; single dose or multiple dosing for 7, 10, 40, and 60 d; residence time in excreta (tE) or soil (tS): 5, 30, 60, 120, 180, 240, 300, and 360 d; disappearance time in excreta (DT50E) or soil (DT50S): no degradation, 10, 30, and 90 d; ploughing depth (D): 25 cm; and restrictive manuring rate: 170 kg N per hectare. It can be emphasized here that the animal data in Table 1 and the agricultural data in Table 2 represent the documentation as assembled by mid-1995. In particular, the recommendations for fertilizer input are subject to change, even on a yearly basis. Therefore, it is advisable to check the validity of the limits whenever a PEC calculation is applied for a certain Member State. Using the basic calculations one could determine what the worst-case PEC value would be. Table 3 presents the results

of these calculations. There are substantial differences in PEC depending on the animal species considered. For a single dose, the highest PECs are estimated for slaughter calves and broilers, intermediate PECs for piglets, and the lowest PECs for dairy cows. A principal reason for this is the number of animals raised per place (N, Eqn. 1), which means that in 1 year nine treatments are given to the broilers but only one for the dairy cows. Other parameters inuence the result in a less predictable way. For example, the difference between dairy cows and slaughter calves, though the same species, is striking but can be explained by examining the data on their body weight and yearly production of nitrogen (Table 1). Per kg body weight, dairy cows produce about 2.3 times more nitrogen (PN /BW 0.155) than calves (PN /BW 0.068 kg). This allows a higher rate for eld application of manure of slaughter calves, i.e., a higher AN/PN ratio (Eqn. 3). In combination with the fact that four treatments are given per slaughter calf place results in the difference in PEC of about nine times in Table 3. The linear relationship between the PEC and the dose rate or the duration of the dosing (Eqn. 1) is also illustrated in Table 3. The 7 d for broilers and 10 d for piglets are typical for a medicated feed, whereas the 40 d for broilers and the 60 d for piglets correspond to a full life cycle treatment as with feed additives. The estimated PEC values indicate that for this type of drug administration, the 10- g/kg limit of concern in soil, as imposed in guidance documents for environmental assessments [1,2], most likely will be exceeded, even for daily doses as low as 1 mg/kg, in practice roughly corresponding to about 10 mg per kg feed. But for single dosing too, as will be the case for many VMs, 10 g/kg is already exceeded in slaughter calves and broilers and approached in piglets, when given at 5 mg/kg, although again, this is not an excessively high dose. Therefore, it seems that a lot of VMs might have to undergo the more elaborate phase II investigations and assessment [2], which contrasts to the original objectives of the phase I [1]. If the worst-case PEC values are exceeding the trigger of 10 g/kg and hence these concentrations are of concern, the PEC could be rened by using the additional equations included in the modied calculations. However, to be able to use these modications, elaborate and expensive additional testing is often required. In Table 4 the inuence of degradation in manure storage (Eqn. 12) as well as after incorporation into soil (Eqn. 13) is addressed. The attenuating factor FD from Equation 14 was calculated for some combinations of residence times and dis-

The PEC of residues of veterinary medicines in soil Table 4. Results of PEC calculations for various disappearance times in excreta (DT50E) or soil (DT50S) and various residence times in excreta (tE) or soil (tS)

Environ. Toxicol. Chem. 16, 1997

1981

DT50E DT50S (d)

10 10 30 30 90 90 90 90 90 90 90
a

tE tS (d)
NRb 5 30 30 180 30 60 120 180 240 300 360

FD
1 0.707 0.125 0.500 0.016 0.794 0.630 0.397 0.250 0.157 0.099 0.063

PEC ( g/kg) 16.07 11.37 2.01 8.04 0.25 12.76 10.13 6.38 4.02 2.53 1.59 1.00

formation is converted to a t/DT50 ratio of 3.3. Thus, as long as the residence time is about three times the disappearance time and, of course, remains in the order of magnitude of normal storage times or agricultural seasons, the disappearance time of the VM should be acceptable. But even for longer disappearance times, kinetic theory indicates that repeated introductions will result in calculable steady-state maximum levels after a number of applications and not in innitely increasing concentrations. For example, with Equation 16 it can be demonstrated, for residues with a DT50S of 180 d and repeatedly introduced on the same area of land with an interval of 1 year, that the maximum concentrations obtained immediately after a new introduction do not increase beyond the fourth or fth introduction. They reach a maximum of about 130% of a single input. This maximum attainable concentration might be a very valuable parameter for environmental risk assessments of long time exposure.
CONCLUSION

The calculation was done for a piglet (BW 20 kg; N 6 per year) receiving a single dose of 10 mg/kg body weight. The manuring was restricted to 170 kg N per year and per hectare. The ploughing depth was 25 cm. FD gives the attenuation factor caused by degradation in manure storage or in soil. b NR, not relevant.

appearance times. The PEC values are for a single dosing of piglets at 10 mg/kg. When transformation in manure as well as in soil is taken into account to estimate the PEC, the attenuation factor is applied twice, although most likely with different values for t and DT50. The PEC of substances with a short DT50, either in manure or in soil, rapidly decreases. From the examples in Table 4, 87.5% is lost within 1 month for a DT50 of 10 d and almost 99% is lost within half a year for a DT50 of 30 d. To some extent this justies the option in the phase I of the draft EU guidelines to exclude from further consideration those substances with a DT50E 30 d. On the other hand, substances with a slower degradation rate do not necessarily persist for a long time at concentrations above the 10- g/kg reference value. For example (Table 4), it takes about 2 months for a substance with a DT50 of 90 d to decrease to the 10- g/kg level from an initial PEC of 16 g/kg. After 180 d, 75% is lost and more than 90% after 300 d. Storage periods of up to 1 year seem realistic with respect to existing regulations to restrict manure spreading to dened periods in the year to allow for immediate ploughing. Manuring intervals on the same area of land in many cases will be at least 1 year and in some cases even several years, to be in accordance with agricultural practice. As a consequence, considerable time might be available in manure storage or in soil for decline of the drug residues. It therefore seems that setting a manure DT50 decision parameter in phase I at 30 d is too conservative and probably redundant when applied in addition to the 10- g/kg limit. The length of the residence time relative to the disappearance time as it enters in Equation 14 seems more relevant. According to Cowan et al. [5], more than 90% of a substance should disappear between two subsequent introductions into the environment to avoid an increase of the PEC. This seems suitable for the residues of VMs in soil too. In the case of annual applications at the maximum permissible level, a halflife of approximately 100 d would still result in 90% depletion and thus no appreciable increase of the PEC. Also for manure storage, a 90% transformation could be suggested as an alternative decision parameter. With Equation 14, the 90% trans-

This paper presents a non-product-specic technique to estimate the PEC of VMs and their residues in soil. It is based on the use pattern of the drug, on physiological parameters of the target animals, on existing regulations on manuring practices, on animal husbandry practices, and on the kinetics in the animal in stored manure or in soil. It offers a uniform calculation routine and a relevant data set, the application of which to various substances may produce comparative information. In the absence of a common approach, data generated individually can be subject to a wide variation and therefore of less value for assessing environmental risk. The output of these calculations in turn can be used as the input for further modelling of the environmental behavior, such as the transport to aqueous environments by leaching or runoff. It essentially applies to the spreading of manure on land that can be ploughed or left undisturbed. At present, there is no eld validation of the model. This was not the original intention either. The model rather should serve as a regulatory screening instrument allowing identication of VMs with a high potential exposure to the environment. When for some substances eld data become available in the future, the validity of the model might be evaluated. The calculation routine will be made available to interested parties as independent computer software, where the user can introduce the substance-specic parameters, which will be combined with the information in the database to produce the PEC estimate. It is hoped that the use of this information and equations will result in a uniform approach in determining the PEC of VMs and their residues in soil and hence contribute to a more transparent and scientic environmental risk assessment of VMs in the EU and worldwide.
AcknowledgementThe authors express their gratitude to Jan Linders of the National Institute of Public Health and the Environment, the Netherlands, to Carol Aldridge of the Veterinary Medicines Directorate, UK, and Andy Croxford of the UK Department of the Environment for their constructive contribution to the calculation model and the nal manuscript.
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3. 4. 5.

6. 7.

8.

9.

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