nature of pesticide residues in fish

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EUROPEAN COMMISSION HEALTH & CONSUMER PROTECTION DIRECTORATE-GENERAL Safety of the food chain Chemicals, contaminants, pesticides

SANCO/11187/2013 31 January 2013 rev. 3

Working document on the nature of pesticide residues in fish.

COMMISSION STAFF WORKING DOCUMENT – DOES NOT NECESSARILY REPRESENT THE VIEW OF THE COMMISION SERVICES

This document has been conceived as a working document of the Commission Services, which was elaborated in co-operation with the Member States. It does not intend to produce legally binding effects and by its nature does not prejudice any measure taken by a Member State within the implementation prerogatives under Regulation 1107/2009 or Regulation 396/2005, nor any case law developed with regard to these provisions. This document also does not preclude the possibility that the European Court of Justice may give one or another provision direct effect in Member States.

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Contents

Abbreviations/Definitions 3

1. Introduction 4

2. Objectives 5

3. Formation of metabolites in fish 5

4. Extent of data required 6

5. Experimental design and reporting requirements 7

5.1. Test substance 7

5.2. Test animals 8

5.3. Housing conditions 8

5.4. Treatment 9

5.4.1. Stability of fortified feed 9

5.4.2. Particularities concerning plateau level 9

5.4.3. Calculation of dietary burden/dose rate 10

5.4.4. Calculation of dose rate 10

5.5. Sampling 11

5.6. Analysis 11

5.7. Strategy for determining when identification of metabolites is needed 14

5.8. Non-extractable residues 15

5.9. Storage stability 17

6. Interpretation 18

7. References 20

Table 1 Strategy for Identification and Characterization of Extractable Residues from

Metabolism in Fish 22

Figure 1 Evaluation scheme non-extractable residues 23

Annex 1 Prediction of the time required to reach steady-state tissue concentrations

(according to OECD TG 305) 25

Annex 2 Feedstuffs Table 28

Annex 3 Dietary burden calculation 30

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Abbreviations/Definitions

ASE Accelerated solvent extraction

BCF Bioconcentration factor

BMF Biomagnification factor

CC Carbohydrate concentrate

CL Crude lipids

CP Crude protein

DM dry matter

GC-MS gas chromatrography/mass spectrometry

HPLC high performance layer chromatography

HR highest residue

IFN Code International Feed Nomenclature codes

IUPAC International Union of Pure and Applied Chemistry

LC-MS liquid chromatrography/mass spectrometry

Log Pow n-octanol/water distribution coefficient

MRBD Maximum reasonably balanced diet

NER non extractable residues

NfE Nitrogen free extract

NMR Nuclear magnetic resonance

NOEC No Observed Effect Concentration

OECD Organisation for Economic Co-operation and Development

PC Protein concentrate

SFE Supercritical fluid extraction

STMR Supervised trials median residue

STMR-P Supervised trials median residue in processed commodity

TLC thin layer chromatography

TRR Total terminal residue

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1. Introduction

Uptake of pesticides by fish, leading to the occurrence of residues in fish products, can occur

following ingestion of feed containing a pesticide residue. Consequently residues in products of

fish origin need to be evaluated.

Fish metabolism data quantify total residues and characterise the chemical nature of residues

which may occur in edible tissues of fish exposed to pesticides. They are required when pesticide

use may lead to significant residues (generally considered to be > 0.1 mg/kg 1of the total diet (dry

weight basis) in fish feed).

A fish metabolism study using 14C radiolabelled test substance should primarily identify the

definition and expression of the residue, and the distribution of residues. Hence analytical

methods are needed to determine residues in fish matrices. A fish metabolism study should also

elucidate the efficiency of extraction of the various components of the residue so that

extraction/residue release procedures can be developed as part of the analytical methods.

The guidance should only be used in connection with residues in fish feed. The direct treatment

of water bodies or spraydrift/run-off/drainage after treatment along water bodies and the possibly

resulting fish exposure is not within the scope of the guidance.

Application

This guidance document is applicable as from 1 January 2014 for all active substances for which

an application for approval and all plant protection products for which an application for

authorisation is made. Transitional measures which are set out in Article 2 of Regulations

amending Regulations (EU) No 544/2011 and No 545/2011 apply to this guidance document.

1 This proposal is not in line with the adopted Regulation on new data requirements which will change the trigger to

0.004 mg/kg bw/d for livestock feeding / metabolism studies. Due to the fact that fishes grow faster than goats and

that the amount of feed has to be adjusted with time it is proposed to leave the trigger as it stands.

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Monitoring results for different fish species are available in Germany from 2001, 2005, 2008,

2010, and 2011. In these analyses persistent organic substances (beneath dioxines and PCBs) like

chlordane, DDT, dieldrin, lindane, toxaphene, HCB, mirex, and bromocyclen were found. Known

active substances found: mercury, copper (heavy metals), chlorpyrifos pendimenthalin, trifluralin,

ethoxyquin (also feed additive). Residues are normally low.

On the basis of these results the guidance shall be applied to all active substances that are fat

soluble, i. e. substances with log Pow ≥ 3.

2. Objectives

The objectives of these studies are:

to provide an estimate of total terminal residues in edible fish commodities;

to determine the distribution of residues;

to identify the major components of the total terminal residue in edible fish commodities;

to quantify the major components of the residue and to show the efficiency of extraction

procedures for these components;

to elucidate the metabolic pathway(s);

to generate data from which a decision on the need for fish feeding studies can be made;

to decide on the definition and expression of a residue for both risk assessment and

enforcement.

3. Formation of metabolites in fish

Ingestion of pesticides and their metabolites present in fish feed may result in uptake and

metabolism of the ingested compounds. Residues in fish will be available as parent compound,

free metabolites, conjugates and/or unextractable / bound residues. Free metabolites (Phase 1)

may be formed by hydrolysis, oxidation, reduction or enzyme-catalysed re-combination.

Conjugates (Phase 2 metabolites) may be formed by reaction of primary metabolites (exocons)

with substances occurring naturally in fish (endocons). Conjugated compounds are usually more

polar than “Phase I metabolites” and dissolve in water or other polar solvents. Conjugates can be

formed with glucuronic acid, glutathione, amino acids and other natural components.

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The exocon (the metabolite of the active substance), as well as the parent compound, can also be

bound covalently to insoluble components. According to IUPAC “a xenobiotic bound residue is a

residue which is associated with one or more classes of endogenous macromolecules. It cannot be

disassociated from the natural macromolecule using exhaustive extraction or digestion without

significantly changing the nature of either the exocon or the associated endogenous

macromolecule”.

In many cases residues that are not readily solubilised in solvents at room temperature have not

been fully defined as “bound residues”. They should then be termed as 'unextractable' or 'non-

extractable' and the procedure used should be specified (Skidmore et al., 1998). It is desirable to

conduct investigations to define the nature of the binding of xenobiotics in food and feed items

and to develop correlation with bio-availability.

More extensive degradation may lead to the release of carbon dioxide or other low-molecular

weight fragments (e.g. formate and acetate). In some cases, these fragments are incorporated into

naturally occurring substances which are of no toxicological concern.

4. Extent of data required

The potential of pesticide residues to accumulate in fish tissue is determined to a significant

extent by the lipophilicity of the active substance (expressed as the n-octanol-water partition

coefficient, POW). The accumulation of compounds of relatively low lipophilicity (log POW < 3)

via the diet is known to be negligible as far as reported residues in fish are taken into account.

Fish metabolism studies are therefore required only for active substances where the log POW is

greater than or equal to three. If a plant metabolite accounts for a major portion of the TRR on a

feed item or if a unique plant metabolite is observed, a fish metabolism study involving dosing

with the plant metabolite may be needed. In these cases the requirement of a fish metabolism

study depends on log POW of the metabolite. Applicants are advised to consult the appropriate

regulatory authority if guidance is needed on the appropriate active ingredient.

Studies should be carried out with one species of fresh water fish of a size appropriate to

commercial food production. Important aquaculture species reared for human consumption such

as rainbow trout (Oncorhynchus mykiss) or common carp (Cyprinus carpio) are the

recommended species (Schlechtriem et al., submitted for publication). These species were chosen

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here as they represent inland aquaculture and different diets (omnivorous vs. carnivorous), and

are easily reared under laboratory conditions.

5. Experimental design and reporting requirements

5.1. Test substance

Experiments should be carried out with radiolabelled substances, preferably 14C, although 35S, 32P

and other radioisotopes may be more appropriate in some cases. As animals are living in water

and tritium is known for its potential for hydrogen exchange, this isotope should not be used. If a

potentially labile side chain is chosen, a metabolism study will be considered adequate only if all

significant radioactivity in the animal is identified and found to be associated with the pesticide,

and not related to loss of the label from the basic structure of the pesticide molecule.

The substance should be labelled in a sufficiently stable position in the molecule to allow all

significant degradation products to be tracked and characterised. Aromatic rings, other cyclic

systems and atoms with several substituent groups usually provide suitable positions for

labelling. Reports must state: the position of radiolabelling, the radiochemical purity (desirable

>95%) and the specific activity.

If an active substance contains multiple ring structures or significant side chains it may be

necessary to conduct separate experiments using two different radiolabels; e.g., labelling

synthetic pyrethroids on either side of the ester bond, or active substances with one phenyl ring

and one heterocyclic ring in both rings etc. A scientifically sound justification might be presented

for using only one radiolabel (e.g.if no cleavage is anticipated).

Occasionally, a fish metabolism study may need to be conducted with a metabolite rather than the

parent active substance. If a plant metabolism study and/or supervised field residue trial indicates

that residues in feed comprise negligible amounts of the active substance, but contain a major

metabolite, which also has a logPow >3 and a fish dietary intake >0.1 mg/kg, then this metabolite

should be used as the test substance in the fish metabolism studies. Although information on the

lipophilicity (log Pow) of the specific metabolite may not always be available, a prediction of its

fat solubility could be supported by information derived from the livestock metabolism studies

(see chapter "Fat-soluble chemicals and additional considerations" in OECD Guideline 505).

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5.2. Test animals

Healthy fish demonstrating normal feed consumption and behaviour should be the appropriate

test animals in the study. Fish may be either all male or all female, or a mix of gender may be

used. Individual fish should be of similar size/weight at the beginning of the dosing phase.

During the study the fish should be observed regularly and any changes in health should be noted

and reported. Reports should specify species and bodyweight at the commencement of dosing and

time of slaughter. Fish selected for the study should have a weight of a minimum of 250 g at the

onset of dosing. It is recommended that a minimum of five fish should be dosed to ensure

sufficient material for analysis. Mortalities exceeding 20% must be justified within the report.

5.3. Housing conditions

Fish should be acclimatised to the experimental conditions for at least two weeks prior to dosing.

During this period fish will be maintained on the unfortified diet and monitored for health,

feeding rate and growth rate. The unfortified diet must be nutritionally balanced to ensure optimal

growth (refer to Table A3.2). Reports should state: date of study, location and conditions (i.e..

water source and chemical properties, water temperature, water exchange rate, dissolved oxygen,

pH, light regime) of basin/tank.

A flow-through test system that provides a sufficient volume of dilution water to the test tanks

should be used to avoid the accumulation of pesticide residues excreted by the test animals into the

test water (Schlechtriem et al., submitted for publication). The flow rates should be recorded.

Suitable filtration systems are recommended to avoid the accumulation of dissolved test item and

metabolites in recirculated water. Water contaminated with radioactive substances must be

disposed of in accordance with the local regulations. The capacity of the tank should be in

compliance with a maximum loading rate of 1 g of fish (wet weight) per litre of water per day.

The filter in used filter systems should be controlled and changed as necessary. The frequency of

changes depends on the type of filtration system used.

The water temperatures should be in the range of 20-25°C for common carp and of 13-17°C for

rainbow trout, respectively. A 12 to 16 hour photoperiod is recommended. Details of lighting

should be reported.

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5.4. Treatment

Owing to the practical difficulties associated with oral dosing of individual fish with

radiochemicals contained in capsules, it is recommended that the dose should be administered via

commercial fish feed (floating and/or slow sinking pelletized diet) that has previously been

fortified with the radiochemical. The feed should have a uniform pellet size to increase the

efficiency of the feed exposure and should be appropriate for the size of the fish used. Using a

radiolabelled test substance avoids the need to confirm that the test substance is otherwise absent

in the diet (i.e. where the test substance is already commercially available). The time taken by the

fish to consume the feed should be reported. If any food remains in the experimental tank 10

minutes after feeding, it should be removed from the tank. The tank should be cleaned 60 minutes

after feeding to remove faeces. Food and faeces are removed to avoid recontamination of the

water with the radiolabelled substance.

5.4.1. Stability of fortified feed

When spiking the feed with the test substance, all possible efforts should be made to ensure

homogeneity throughout the test feed. Fortified feed pellets should be stabilized by surface

coating (e.g. corn oil or sodium alginate) to avoid leaching losses prior to the ingestion of the

experimental diet (Goeritz et al., submitted for publication). The methodology for preparing the

dose and the extent of leaching from the treated pellets should be described in the report.

Leaching losses should not exceed a maximum of 10% prior to ingestion of the daily ration.

5.4.2. Particularities concerning plateau level

The duration of the uptake phase should be calculated prior to the experiment, to confirm the

anticipated worst case time to reach 95% plateau according to the equations given in Annex 1.

Ideally dosing should be continued until tissue concentrations are predicted to reach a steady-

state. However, in practice dosing should last for no longer than 10-14 consecutive days due to

high experimental inputs. In cases of highly lipophilic substances (e.g. log POW ≥ 5) the uptake

phase should be terminated after 10-14 days and the steady-state concentrations in the tissues

extrapolated from BMF data estimated in a dietary test according to OECD TG 305 (2012) (refer

to Annex 1 for further advice). However, BMF data expresses the total accumulation potential of

dietary contaminants in the entire fish. Therefore, information on the distribution of the test item

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in the fillet compared to entire carcass is required to deduce fillet-specific steady-state

concentrations.

5.4.3. Calculation of dietary burden/dose rate

The calculation of the dietary burden is based on the specific highest residue (HR) or median

residue values (STMR, STMR-P) as appropriate for the feed commodities. These values are

derived from supervised crop residue trials. Feed commodities relevant for fish feeding and their

maximum inclusion rates for aquaculture diets are given in the Feedstuffs Table (Annex 2). The

classification of the feed commodities is represented by carbohydrate concentrate (CC; contains

<30% protein), protein concentrate (PC; contains >30% protein) and fat (F). The dietary burden will

be calculated for common carp and rainbow trout based on the formulation of Maximum

Reasonable Balanced Diets (MRBD) and should be expressed in mg/kg feed on a dry matter (DM)

basis (Annex 3) and as mg/kg bw/d. The target composition of MRBD (% of DM) is 42% and

35% crude protein and 15% and 10% crude fat for rainbow trout and Common carp, respectively.

Hand formulation of MRBD is possible according to the principles described in Annex 3, but will

lead only to an approximation of the dietary burden. In practice, the MRBD should be calculated by

linear programming to optimize the dietary burden estimates.

5.4.4. Calculation of dose rate

The daily dosage of fish must at least match the maximum daily exposure based upon the

calculation of dietary burden (Annexes 2 and 3). In practice there may be analytical problems if

dose rates of less than 10 mg/kg feed DM are used. If necessary for identification of metabolites

administration of higher doses may be useful. However, the test substance’s toxicity (NOEC if

known) should be always taken into account. Fish are fed at a fixed ration following the feeding

recommendations provided by the food manufacturer (approximately 3% of wet body weight per

day). The exact feeding rate and amount of feed applied during the experiment must be recorded.

The feed ration should be adjusted after 50% of the intended experimental period is completed to

account for the expected growth increment of the animals. Expected growth can be calculated

based on food conversion ratios usually stated for most commercial fish diets. More thoroughly,

the expected growth of fish should be determined in feeding pre-tests prior to the fish metabolism

study. Weighing of the fish during the study should be avoided because any disturbance of the

animals may affect the ingestion of the experimental diet. Feeding must be constantly observed to

ensure that the fish are visibly consuming all of the food presented. If food is consistently being

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left uneaten, it may be advisable to spread the dose over an extra feeding period in the feeding

schedule (e.g. feeding half the amount twice daily). Faeces should as far as possible be removed

from the tank. The acceptability of the experimental diet should be tested in advance with non-

radiolabelled material to make sure it is palatable. Reports should state: dose rate, number and

timing of treatments, method of dosing and total feed consumed as well as the weights of

individual fish at start and end of dosing phase.

Due to the specific experimental conditions applied during a fish metabolism study the

determination of a mass balance is of limited value and thus not required.

5.5. Sampling

The experimental animals should be exposed to the fortified diet until the end of the uptake

period. Experimental animals should be slaughtered 6-12 hours after the last feeding event. The

total weight of each carcass should be recorded. The tissues to be sampled are muscle (fillet)

including skin and liver tissue. Tissue samples should be dissected and the weight of each tissue

recorded in the raw data. If the samples cannot be analysed immediately, they should be stored

frozen at or below –18°C.

Reports should state: nature, number and size/weight of samples taken. Storage duration and

temperature prior to analysis should be stated.

5.6. Analysis

In the analytical phase of a fish metabolism study, the animal parts to be analyzed are sampled,

chopped or homogenized and the total radioactive residue (TRR) determined. Full accountability

of all radioactivity must be ensured. The study should assess TRRs in muscle/skin (fillet) tissue

(combined sample containing muscle and skin in natural proportions) for food safety purposes,

and if possible, in liver tissue to establish a biotransformation pathway for the active substance.

Tissue samples of the same type from individual fish may normally be pooled prior to initial TRR

analysis. Validation of the extractability of residues may be required in some cases to support the

results obtained.

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Samples are extracted with a series of solvents and/or solvent systems (including aqueous) with

various polarities and other characteristics depending on the nature of the expected residues.

These initially obtained residues are defined as extractable residues. The requirements for

characterization and/or identification of extractable residues are summarized in Table 1.

The pathway described below should be viewed as a broad outline of the type of information

needed to determine that a fish metabolism study is acceptable. Different procedures and

methodologies may be appropriate in a given circumstance. The basic concepts regarding

"trigger" values for identification of radioactivity, methodologies required for characterization

and/or identification of radioactivity, and steps that should be taken to assure adequate release of

unextractable residues should be observed to assure that the submitted study is adequate. These

trigger values (see Table 1) are meant as rough guidance and may not apply to situations where a

metabolite is suspected to be of particular toxicological concern, or where less than 10% of the

TRR represents a high absolute residue level.

The lipid content of the homogenized muscle samples should be analysed to allow normalisation

of the measured concentrations to average lipid content, if required. Suitable methods should be

used for determination of lipid content. The chloroform/methanol extraction technique, according

to Bligh and Dyer (1959) or the Smedes (1999) method using non-chlorinated solvents, should be

used as explained by Schlechtriem et al. (2012).

Identification refers to the exact structural determination of components of the total radioactive

residue. Typically, identification is accomplished either by co-chromatography of the metabolite

with known standards using two dissimilar systems or by techniques capable of positive

structural identification such as MS, NMR, etc. In the case of co-chromatography,

chromatographic techniques utilizing the same stationary phase with two different solvent

systems are not considered to be an adequate two-method verification of metabolite identity,

since the methods are not independent. Identification by co-chromatography should be obtained

using two dissimilar, analytically independent systems such as reverse and normal phase thin

layer chromatography (TLC) and high performance layer chromatography (HPLC). Provided that

the chromatographic separation is of suitable quality, then additional confirmation by

spectroscopic means is not necessary. Unambiguous identification can also be obtained using

methods providing structural information such as: liquid chromatography/mass spectrometry

(LC-MS), or liquid chromatography/tandem mass spectrometry (LC-MS/MS), gas

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chromatography/mass spectrometry (GC-MS), and NMR. If the metabolite is determined to be of

minimal importance due to its low absolute level (less than 0.05 mg/kg tissue) or percentage of

the TRRs (less than 10% of the TRRs), identification by co-elution with putative synthetic

metabolites as reference standards using one chromatographic technique e.g., reverse phase

HPLC, will be acceptable.

Characterization refers to the elucidation of the general nature/characteristics of the radioactive

residue short of metabolite identification. Terms used to characterize residues include

organosoluble, water or aqueous soluble, neutral, acidic or basic, polar, nonpolar, nonextractable,

etc. Characterization may also involve descriptions of chemical moieties known to be present in

the molecule based on conversion to a common structure or due to reactivity with particular

reagents. The degree of characterization refers to how close the assignment comes to structural

identification.

When identification of radioactive residues is not accomplished, the degree of characterization

required for a portion of the total radioactivity will depend on several factors including the

amount of residue present, the amount of the TRR already identified, the toxicological concern

over a class of compounds, the suspected significance of the residue as determined by

characterization already performed, the results obtained in other livestock metabolism studies and

the capability of analytical methods to detect characterized but unidentified residues.

In the metabolism studies in which highly exaggerated feeding levels are employed and low

radioactivity results in tissues, characterization and/or identification requirements should be less

stringent than when the expected dietary burden already leads to significant radioactivity in fish

products. For example, if the anticipated dietary burden to fish is about 0.1 mg/kg dry feed,

10 mg/kg radiolabelled compound (dry weight basis) is fed (100X), and total radioactivity in

tissues is less than 0.01 mg/kg tissue, minimal characterization and/or identification of residues

should be adequate (unless for substances of toxicological concern).

When radioactive residues greater than 0.01 mg/kg tissue are observed in fish commodities from

ingestion of the pesticide at levels expected in feed items, thorough identification of the residues

is generally necessary. This is likely when pesticides are applied to crops at high rates late in the

growing season. The procedure for identification of residues outlined in Table 1 should be

followed.

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New extraction and analysis techniques may be appropriate as a substitute for the techniques

mentioned above. Alternate extraction procedures such as supercritical fluid extraction (SFE),

microwave extraction and accelerated solvent extraction (ASE) can be used. In any case, the best

available technique should be used to fully elucidate the metabolic pathway.

Reports should include: full details of the methods of analysis (including extraction scheme),

recoveries of radioactivity in relation to the measured radioactivity in the specific tissue, and

sufficient representative chromatograms, spectra and photographs of auto-radiograms to illustrate

the quality of the data and to indicate the confidence which can be attached to characterisations

and identifications.

5.7. Strategy for determining when identification of metabolites is needed

The threshold values shown in Table 1 reflect the minimum level of characterization and/or

identification required for each tissue or organ following application of the radiolabelled test

compound. If the TRR in an animal commodity is 0.01 mg/kg or less, no differentiation of

compounds would be necessary unless toxicological concerns over residues occurring at lower

levels have been identified. For TRR greater than 0.01 mg/kg, the sample should be extracted

with solvents or solvent mixtures of various polarities. The components of the extractable

radioactive residue (parent compound and metabolites) should then be quantitated by

chromatographic analysis (TLC, HPLC) to determine the degree of characterization that is

needed.

If the extractable TRR represents 0.01 mg/kg tissue or less, it does not need to be examined

further. For extractable TRR of 0.01-0.05 mg/kg tissue, the chromatographic behaviour of this

residue can be compared to that of the parent pesticide and likely metabolites (characterisation

and/or identification).

When the extractable radioactive residue exceeds 0.05 mg/kg or 10% of the TRR, whichever is

greater, characterization and identification should be attempted in each fraction. The exception

for this would be toxicology concerns over potential residues which might occur at lower levels.

Low levels (in terms of both mg/kg and % of TRR) of individual residues do not typically need to

be identified if the major components of the residue have been identified. For example, if the total

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radioactive residue in a tissue (or organ) is 3 mg/kg and 75% of that has been firmly identified, it

is unlikely that identification of a series of individual residues in the 0.05-0.1 mg/kg range would

be required. On the other hand, extensive efforts towards identification of 0.05-0.1 mg/kg

residues would be expected when the total radioactive residue is only 0.3 mg/kg. It should be

noted that the trigger values (on a concentration basis) are not absolute standards, but intended to

provide guidance as to how much characterization is adequate. However, in many cases, a

potentially important metabolite may partition into multiple fractions because of solubility

characteristics, and/or because it is present in both free and conjugated forms. In order for the

trigger values to apply, particularly in cases where the TRRs are distributed among numerous

fractions, it should be demonstrated (e.g., by chromatographic analysis of each fraction) that no

single metabolite is distributed among the various fractions in such amounts that the combined

level (sum) of this component significantly exceeds the trigger value.

The relative amounts of radioactive residues, shown in Table 1, apply regardless of the dose rate

used in fish metabolism studies. If doses less than 10 mg/kg feed DM are used, this is often

insufficient to provide adequate absolute amounts for characterization and/or identification of

residues, additional studies will be needed to provide sufficient amounts of radioactive residues

by appropriate means, e.g., increased specific radioactivity, suitable time of sacrifice or

exaggerated dose.

5.8. Non-extractable residues

There are three situations in which radioactive residues are observed to be non-extractable in fish:

Incorporation into biomolecules (i.e., amino acids, sugars, etc.). This occurs when the test

compound is degraded into small (usually one or two) carbon units which enter the carbon

pool of endogenous compounds used in the biosynthesis of new cell constituents by the

animal.

Chemical reaction or physico-chemical tight-binding with appropriate moieties in

biomolecules to form bound residues, which can be released via other chemical reactions

(e.g., enzymatic or acid/base hydrolysis).

Physical encapsulation (trapping) or integration of radioactive residues into fish matrices.

Release of residues in this situation may require solubilisation of the tissue, usually by drastic

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treatment with base, although use of surfactants may allow the radioactive residue to be

released under less severe conditions.

The following general road map for dealing with non-extractable residues is intended to provide

guidance and clarification regarding characterization and/or identification of these residues.

The extracted solid animal material should be assayed and, if the radioactive residue exceeds the

trigger values of 0.05 mg/kg or 10% of the TRR, whichever is greater, release of the radioactivity

should be attempted (see Table 1). It is emphasized that, if toxicologists express concerns over

potential residues at lower levels; the trigger values will not necessarily apply.

At each step in Table 1, the total radioactivity in the released residues should be quantitated. With

respect to characterization, it should be emphasized that the chromatographic behavior of the

released radioactive residue (including water soluble materials) should be compared to that of the

parent and likely metabolites, which are close in structure to the parent. This will indicate

whether the released radioactive residue is chemically different from the parent molecule. If the

remaining unextracted radioactive residue after a given procedure is less than 0.05 mg/kg or less

than 10% of the TRR, further attempted release of radioactive residues is not necessary.

Treatments may be performed sequentially or on sub-samples. The types of treatments include

addition of dilute acid and base at 37°C (note that these procedures should be employed initially

for both metabolism and method development considerations), or the use of surfactants, enzymes,

and 6N acid and/or 10N base with reflux. It should be kept in mind that the milder procedures

provide more accurate assignments of metabolite structures released, i.e., acid/base reflux would

probably release moieties as their final hydrolysis products, which could have only a minor or

remote relationship to the conjugated non-extractable form of the radioactive residue.

An ambient temperature acid treatment followed by ambient temperature base treatment will

provide a mild hydrolysis of conjugated moieties, and again possibly release any biomolecules

containing incorporated radioactive residues. The use of surfactants may release physically

encapsulated or membrane bound residues. Because membrane and/or cell wall disruption may

improve substrate accessibility to the enzyme, a sonication step should be employed followed by

a carefully chosen enzymatic battery. (Note: In each case the activity of each enzyme utilized

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should be confirmed.) These steps could release chemically-bound residues including any

biomolecules containing incorporated radioactive residues.

The final release steps would involve reflux acid and base hydrolysis, which will likely solubilise

the animal tissue. Radioactive residues released at this time would probably reflect amino acids,

sugars and encapsulated or conjugated compounds, which may or may not have any relationship

to the original non-extractable/encapsulated structures. However, this step does provide evidence

that residues of the pesticide can be released, and may provide data on incorporated radioactive

residues and limited information about the nature of the metabolites. In all cases, samples,

homogenates and extracts should be maintained at low temperatures except during hydrolytic

steps in order to reduce degradation/artefact formation. Figure 1 provides a visual description of

the steps discussed above.

Identification of specific radiolabelled endogenous compounds like amino acids, sugars, phenolic

compounds, nucleotides, etc. may alleviate the need for further characterization and/or

identification of non-extractable residues in many instances, since this usually means that the

pesticide has been degraded into small carbon units which have entered the carbon pool. This

conclusion does not, however, apply to pesticides in which the 14

C label is incorporated at a labile

site in the molecule. This conclusion would also not apply in cases where a single released

metabolite, which comprises a significant portion of the TRR (greater than 10% of the TRR or

greater than 0.05 mg/kg), has not been identified.

5.9. Storage stability

Determinations as to whether sample integrity was maintained during collection, sample

preparation, and storage should be made. Such analyses should show that the basic profile of

radiolabelled residues has not changed throughout the duration of the study.. Storage stability

data is not normally necessary for samples analyzed within six months of collection, provided

evidence is given that attempts were made to limit degradation of residues by appropriate storage

of matrices and extracts during the analytical portion of the study.

If instability of the active ingredient is suspected or observed, based on other information, steps

should be taken to safeguard the integrity of the study. In those cases where a metabolism study

cannot be completed within six months of sample collection, evidence should be provided that

18

the identity of residues did not change during the period between collection and final analysis.

This can be done by analyses of representative substrates early in the study and at its completion.

The substrate should be the item stored, i.e., if the matrix extract is used throughout the study and

the matrix is not extracted later in the study, the stability of the extract should be shown.

If changes are observed (e.g., disappearance of a particular HPLC peak or TLC spot), additional

analyses or another metabolism study with a shorter collection to analysis interval may be

necessary.

Ideally metabolism samples should be stored at/or below –18oC. Storage under any other

conditions needs to be recorded and justified.

6. Interpretation

In some cases a fish metabolism study may be used to indicate that transfer of residues is minimal

(e.g. low lipophilic residues) and that the expected dietary exposure will generate residues less

than the limit of determination in a feeding study at a 1X level. In this case, dosing is required for

a maximum period of 14 days or until plateau levels are reached, whichever period is shorter. Full

scientific justification should be given for the duration chosen (Annex 1). Consequently, a

feeding study that estimates MRLs is unnecessary.

The relevance of results should be discussed in relation to the proposed uses and the expected

dietary burden resulting therefrom. The toxicological significance of metabolites (including non-

extractable residues) should be considered.

In case of non-extractable residues the question remains as to whether such residues will be

available to biological systems, including humans when persisting in certain constituents of feed

or food. If biological unavailability of residues can be demonstrated, such residues can be

considered non-significant. Figure 1 shows an evaluation scheme for non-extractable residues.

It may be necessary to evaluate the bio-availability of conjugated metabolites in the same way as

non-extractable residues. The extractability of residues should be discussed in relation to the

proposed analytical methodology.

19

A schematic diagram of the metabolic pathway with a brief explanation of the distribution and

chemical changes involved should be provided in the report and a residue definition for

enforcement and risk assessment should be proposed.

20

7. References

Bligh E. G., Dyer W. J., 1959: A rapid method of total lipid extraction and purification. Can. J.

Biochem. Physiol. 37: 911-917

Goeritz I., Atorf C., Whalley P., Seymour P., Klein M. Schlechtriem C.: Fish metabolism studies

for regulatory purposes: Investigations on feed preparation. Journal of the Science of Food and

Agriculture: Submitted for publication.

Hertrampf J W, Piedad-Pascual, F., 2000: Handbook on Ingredients for aquaculture feeds,

Kluwer Academic Publishers, Dordrecht, The Netherlands.

Jeroch H., Drochner, W., Simon, O., 1999: Ernährung landwirtschaftlicher Nutztiere. Ulmer

Verlag, Stuttgart, Germany.

Organisation for Economic Cooperation and Development, 2012: Draft TG 305:

Bioaccumulation in fish: Aqueous and dietary exposure, Paris, April 2012

Organisation for Economic Cooperation and Development, 2007: Metabolism in Livestock.

OECD Guideline for the Testing of Chemicals No 503, Paris, January 2007

Organisation for Economic Cooperation and Development, 2007: Residues in Livestock. OECD

Guideline for the Testing of Chemicals No 505, Paris, January 2007

Organisation for Economic Cooperation and Development, 2007: Guidance Document on

Overview Of Residue Chemistry Studies (as revised in 2009). Series on Testing and Assessment

Number 64 and Series an Pesticides Number 32, July 2009.

Schlechtriem, C., Fliedner, A., and Schäfers, C., 2012. Determination of lipid content in fish

samples from bioaccumulation studies: Contributions to the revision of guideline OECD 305.

Environmental Sciences Europe. 24: 13

21

Schlechtriem C., Goeritz, I., Atorf C., Bergendahl E., Seymour P., Whalley P.: Development of a

regulatory testing procedure to study the metabolism of pesticides in farmed fish. Pest

Management Science: Submitted for publication.

Skidmore M. W., Paulson G. D., Kuiper H. A., Ohlin B. and Reynolds S., 1998:

Bound Xenobiotic Residues in Food Commodities of Plant and Animal Origin. IUPAC Reports

on Pesticides(40), Pure & Appl. Chem. 70, 1423, 1998

Skidmore M.W., 2000: Bound residues arising from the use of agrochemicals on plants. In

Metabolism of Agrochemicals in Plants, Edited by T. Roberts, John Wiley & Sons LTD,

Chichester, 2000.

Smedes F., 1999: Determination of total lipid using non-chlorinated solvents. Analyst 124: 1711-

1718

Bligh E. G., Dyer W. J., 1959: A rapid method of total lipid extraction and purification. Can. J.

Biochem. Physiol. 37: 911-917

Tacon A. G. J., 1993: Feed ingredients in for warmwater fish. Fishmeal and other processed

feedstuffs. FAO Fisheries Circular No. 856, FAO, Rome.

Hertrampf J W, Piedad-Pascual, F., 2000: Handbook on Ingredients for aquaculture feeds,

Kluwer Academic Publishers, Dordrecht, The Netherlands

Jeroch H., Drochner, W., Simon, O., 1999: Ernährung landwirtschaftlicher Nutztiere. Ulmer

Verlag, Stuttgart, Germany.

22

Table 1 Strategy for Identification and Characterization of Extractable Residues from Metabolism in Fish

Relative amount

(% TRR)

Concentration (mg/kg) Required Action

< 10 < 0.01 No action if no toxicological concern.

< 10 0.01 – 0.05 Characterize, only attempt to confirm identity if

straightforward (e.g., a reference compound is

available or the identification is known from a

previous study).

< 10 > 0.05 Characterization/identification needs to be

decided on a case-by-case basis taking into

account how much has been identified.

< 10 < 0.01 Characterize. Only attempt to confirm identity if

straightforward (e.g., a reference compound is

available or the identification is known from a

previous study).

> 10 0.01 – 0.05 Significant attempts to achieve identification

should be made especially if needed to establish a

pathway, ultimately characterization might be

accepted.

> 10 > 0.05 Identify using all possible means.

> 10 > 0.05 Unextracted

radioactive residue

Unextractable radioactive residue – See Figure 1.

23

Figure 1 Evaluation scheme non-extractable residues

Unextracted radiolabel

No additional characterization(toxicological consideration

permitting)

6N acid and/or10N base reflux

Enzymes

Surfactants orultrasonication

Dilute acid and/orbase (ambienttemperature)

Final hydrolysisproducts of

pesticide

Labelled sugars, aminoacids, etc., Labelled

conjugates of sugars,amino acids, etc.

Physicallyencapsulatedmetabolites

Metabolites andconjugates

Levels down to 0.05 mg/kg or 10%of the TRR, whichever is greater

Sonication

< 0.05 mg/kg or< 10% of the TRR, orwhichever is greater

Comments on Table 1 and Figure 1:

At each step in Figure 1, the radioactivity of the released residues should be quantitated. If

the trigger values shown in Table 1 for extractable residues are met, the radioactive

residue should again be partitioned against various solvents/solvent mixtures and

characterized and/or identified as required. With respect to characterization, it should be

emphasized that the chromatographic behaviour of the released radioactive residue

(including water soluble materials) should be compared to that of the parent and likely

metabolites, which are close in structure to the parent. This will indicate whether the

released radioactive residue is chemically different from the parent molecule. If the

remaining unextracted radioactive residue after a given procedure is less than 0.05 mg/kg

or less than 10% of the TRR, further attempted release is not necessary.

24

The trigger values shown in Table 1 are meant to negate the need for characterization

and/or identification of metabolites present at very low and insignificant levels. However,

in many cases, a potentially important metabolite may partition into multiple fractions

because of solubility characteristics, and/or because it is present in both free and

conjugated forms. In order for the trigger values to apply, particularly in cases where the

TRR is distributed among numerous fractions, it should be demonstrated (e.g., by HPLC

analysis of each fraction) that no single metabolite is distributed among the various

fractions in such amounts so that the combined level (sum) of this component

significantly exceeds the trigger value.

25

Annex 1 Prediction of the time required to reach steady-state tissue concentrations (according to OECD TG 305)

The duration of the uptake phase to reach a steady-state during bioconcentration studies can be

predicted based on the bioaccumulation kinetics of the test item. The same approach can be used

when estimating a steady state from a biomagnification factor (BMF) obtained in the dietary test.

In this case, BCF is replaced with BMF and Cw with Cfood, concentration in the food, in the

respective equations.

An estimate of k2 (depuration rate) and hence some percentage of the time needed to reach a

steady-state may be obtained from empirical relationships between k2 and the n-octanol/water

partition coefficient (KOW) or k1 (uptake rate) and bioconcentration factor (BCF). It should be

realised, however, that the equations in this section only apply when uptake follows first-order

kinetics. If this is clearly not the case it is advised to seek advice from a biostatistician, if

predictions of the uptake phase are desirable.

An estimate of k2 (day-1) may be obtained by several methods. For example, the following

empirical relationships could be used:

47.1)(log414.0)(log 10210 +−= owKk (r2=0.95) [equation A1.1]

or

BCF1

2kk =

[equation A1.2]

Where 32.01 W520 −⋅=k (for compounds with a log KOW > 3) [equation A1.3]

And 70.0log85.0BCF OW −⋅= K [equation A1.4]

W = mean treated fish weight (grams wet weight) at the end of uptake/start of depuration

The time to reach some percentage of steady-state may be obtained, by applying the k2-estimate,

from the general kinetic equation describing uptake and depuration (first-order kinetics):

26

f2w1f CkCk

dtdC

−= [equation A1.5]

or, if Cw is constant:

( )tkeCkkC 21w

2

1f

−−⋅= [equation A1.6]

When steady-state is approached (t → ∞), equation A1.6 may be reduced to:

w2

1f C

kkC ⋅=

[equation A1.7]

or

BCF2

1

w

f ==kk

CC

[equation A1.8]

Then BCF × Cw is an approximation to the concentration in the fish at steady-state (Cf,s).

Equation A1.6 may be transcribed to:

( )tkeCC 21f,sf−−⋅= [equation A1.9]

or

tkeCC

21sf,

f −−= [equation A1.10]

Applying equation A1.10, the time to reach some percentage of steady-state may be predicted

when k2 is pre-estimated using equation A1.1 or A1.2.

As a guideline, the statistically optimal duration of the uptake phase for the production of

statistically acceptable data (BCFK) is that period which is required for the curve of the logarithm

27

of the concentration of the test substance in fish plotted against linear time to reach its mid-point,

or 1.6/k2, or 80% of steady-state but not more than 3.0/k2 or 95% of steady-state.

The time to reach 80 percent of steady-state is (using equation A1.10):

802180.0 tke−−= [equation A1.11]

or

2280

6.1)20.0ln(kk

t =−

= [equation A1.12]

Similarly 95 percent of steady-state is:

2295

0.3)05.0ln(kk

t =−

= [equation A1.13]

For example, the duration of the uptake phase (tuptake) for a test substance with log POW = 4 would

be (using equations A1.1, A1.12 and A1.13):

( ) ( ) 47.14414.0log 210 +−=k

k2 = 0.652 day-1

( ) hours) (59 days 45.2652.06.1%80 ==uptaket

or ( ) hours) (110 days 60.4

652.00.3%95 ==uptaket

Alternatively, the expression:

(hours) 31.551054.6 OW3

eq +⋅⋅= − Pt [equation A1.14]

may be used to calculate the time for effective steady-state (teq) to be reached.

28

Annex 2 Feedstuffs Table

Fish in aquaculture are fed based on a “maximum reasonably balanced diet (MRBD)” approach

only. The approach uses fixed percentages of carbohydrate concentrate, protein concentrate and

fat. (Note: This concept will shortly be introduced in dietary burden calculations for livestock for

regions where intensive feeding practices predominate. The respective OECD Guidance

Document on Residues in Livestock is currently under preparation, finalization and submission

are envisaged for 2012). Individual feed commodities are classified into specific feedstuff

categories (forages, roots and tubers, cereal grains/crop seeds, by-products and fat).

Information on the various plant commodities commonly used for the formulation of aquaculture

diets is provided in the following table:

Crop Commodity IFN Code Class

Residue Input value

CP** (%) of DM

NfE (%) of DM

CL (%) of DM DM

(%)

Carp* (max. % of diet)

Trout* (max. % of diet)

Forages/Fodders Dried grass (e.g. Alfalfa meal) is a potential minor ingredient in fish and shrimp feeds as a source of carotenoids. Limited value in fish feeds except for herbivorous fish

Roots and tubers Limited value as ingredients for aquaculture feeds; usually only used in small quantities e.g. as binders increasing the water stability of diets.

Cereal Grains/Crop Seeds Faba bean Treated seed CC STMR 28.3 34.9 8.4 88 15 15

Cow pea Treated seed 5-01-661 CC STMR 25.1 58.7 4.9 88 15 15

Lupin (white) Treated seed 5-02-707 PC STMR 34.5 40.2 6.1 88 15 15

Pea Treated seed 5-03-600 CC STMR 23.7 64.3 1.7 90 15 15

Corn grain 4-20-698 CC STMR 10.6 80.6 2.6 88 35 20

Rice Broken grains 4-03-939 CC STMR 8.1 90.2 0.6 88 50 10

Sorghum grain 4-04-383 CC STMR 11.5 69.1 3.1 86 35 18

Soybean Treated seed 5-64-610 PC STMR 39.8 28.3 20.3 89 40 25

Sunflower seed CC STMR 19.1 49.5 10.8 88 35 15

Triticale grain 4-20-362 CC STMR 14.5 78.7 2.8 88 15 15

Vetch seed 5-26-351 CC STMR 25.2 62.2 1.15 89 15 15

Wheat Grain (extruded) 4-05-211 CC STMR 13.8 79.4 2.9 89 35 20

By-Products

Barley bran fractions 4-00-515 CC STMR-P 16.4 68.0 6.6 88 35 15

Brewer's grain dried 5-00-516 CC STMR-P 25.9 44.5 7.0 92 35 15

Coconut/Copra meal decorticated 5-01-572 PC STMR-P 44.2 28.9 1.2 91 25 15

Corn, field Grain meal 4-12-208 CC STMR-P 10.2 80.6 4.8 87.8 35 20

Corn, field bran 5-28-235 CC STMR-P 15.0 63.7 5.7 87.5 20 5

Corn, field hominy meal 4-03-010 CC STMR-P 8.9 78.8 5.4 90.1 35 20

Corn gluten feed 5-28-243 CC STMR-P 24.7 55.4 3.5 90.1 20 10

Corn gluten meal 5-28-242 PC STMR-P 59.9 31.6 3.6 91.3 20 15

Corn starch 4-02-889 CC STMR-P 0.4 99.0 0.4 90.2 35 15

Cottonseed meal 5-01-617 PC STMR-P 32.9 27.6 1.7 90 35 15

Distiller's grain dried 5-00-518 CC STMR-P 28.5 46.9 10.2 92 10 10

29

Crop Commodity IFN Code Class

Residue Input value

CP** (%) of DM

NfE (%) of DM

CL (%) of DM DM

(%)

Carp* (max. % of diet)

Trout* (max. % of diet)

Leucaena leaf meal CC STMR-P 24.5 51.1 5.4 90.5 10 -

Linseed meal 5-02-048 CC/PC STMR-P 35.0 37.6 2.0 90 35 15

Lupin seed white meal (treated) 5-27-717 PC STMR-P 34.5 40.2 6.1 89.5 15 15

Mustard meal PC STMR-P 42.4 30.3 1.8 89.9 10 10

Olive cake CC STMR-P 13.3 44.5 3.6 92.4 10 10

Palm kernel meal 5-03-486 CC STMR-P 16.3 50.4 1.4 90 10 8

Peanut meal decorticated 5-03-649 PC STMR-P 46.5 29.1 1.0 90.2 35 15

Potato protein PC STMR-P 81.8 12.2 2.8 89.4 3 -

Rape seed meal (toxic) 5-26-093 PC STMR-P 37.3 33.2 1.9 91 5 5

Canola meal 5-08-136 PC STMR-P 37.3 33.2 1.9 91 35 20

Rice Bran de-oiled CC STMR-P 15.1 52.8 1.7 100 35 15

Rice polishing CC STMR 13.6 59.4 14.5 100 50 10

Rice hulls 1-08-075 CC STMR-P 3.1 34.2 1.0 100 5 0

Sesame seed meal 5-04-220 PC STMR-P 45.0 24.2 4.8 92.4 35 15

Safflower Meal decorticated 5-26-095 PC STMR-P 45.2 20.9 6.9 91 7 7

Soybean Meal decorticated 5-20-638 PC STMR-P 49.8 30.1 0.8 89.5 40 25

Soybean protein 5-24-811 PC STMR-P 72 21.0 1.0 92 20 20

Sunflower meal decorticated 5-26-098 PC STMR-P 43.5 28.2 3.2 92.6 20 10

Wheat Extruded grain 4-12-208 CC STMR-P 13.5 80.2 1.9 87.7 15 15

Wheat bran 4-05-190 CC STMR-P 15.6 61.8 4.7 88.7 35 15

Wheat flour 4-05-199 CC STMR-P 14.3 81.9 1.7 88.0 15 15

Wheat germ 5-05.218 CC STMR-P 28.5 54.3 8.8 88.7 5 5

Wheat middlings 4-06-749 CC STMR-P 16.9 66.2 4.4 89.4 40 25

Wheat gluten PC STMR-P 80.1 17.2 1.5 91.4 15 15

Fat

Vegetable oil*** oil F STMR-P 100 10 15

*Recommended maximum inclusion rates are based on Tacon 1993, and Hertrampf & Piedad-Pascual 2000.

**Proximate composition of crop commodities (% dry matter (DM)) according to Hertrampf & Piedad-Pascual and

Jeroch et al. 1999. (CP = crude protein; NfE = Nitrogen free extract; CL = Crude lipids)

***All types of plant (oilseed ) oils which can be used for aquaculture diets such as canola oil, cottonseed oil,

safflower oil, soybean oil, sunflower oil, palm oil, peanut oil, linseed oil, corn oil, coconut oil etc.

IFN Codes stand for International Feed Nomenclature codes, which are listed for most commonly used feedstuffs

from crops.

The classification of the feedstuff is represented by CC: carbohydrate concentrate; PC: protein concentrate;

F: Fat.

Entries for the residue input: Supervised trial median residue (STMR), Supervised trial median residue multiplied by

a processing factor (STMR-P).

% DM represents the % dry matter.

30

Annex 3 Dietary burden calculation

The hand formulation of Maximum Reasonable Balanced Diets (MRBD) for fish is described

here to show the principles of dietary burden calculation. However, this procedure can only

provide an approximation of the dietary burden in aquaculture diets. In practice, the MRBD

should be calculated by linear programming to optimize the dietary burden estimates.

A 3.1 Steps to follow:

Select all feedstuffs (see Annex 2) potentially treated with the test item (pesticide).

List the specific residue value (mg/kg) for each feedstuff. Only blended and/or processed

commodities (grains, byproducts) are used and therefore the respective median residue

from the supervised field trials (STMR, STMR-P) should be applied.

Rank the single commodities according to their contribution to the dietary burden.

Conduct two separate calculations, one for rainbow trout and one for common carp.

A 3.2 Calculation of the dietary burden by formulation of the Maximum Reasonable

Balanced Diet (MRBD)

Select a protein supplement, protein >30% from the list of protein concentrates (PC) with

the highest STMR (STMR-P) value.

Select a basal ingredient, protein <30% from the list of carbohydrate concentrates (CC)

with the highest STMR (STMR-P) value.

Table A3.1 Selection of protein supplement and basal ingredient (Example)

CP (%) CL (%) STMR-P (mg /kg DM) 1.Peanut meal (PC) 46.5 1.0 0.09 2.Soybean meal (PC) 49.8 0.8 0.05 1.Corn meal (CC) 10.2 4.8 0.3 2.Rice (broken grains) (CC) 8.1 0.6 0.02 1.Vegetable oil (F) 0 100 0.01

Calculate a sample, 2 ingredient diet of the target composition (Table A3.2) containing

42% in rainbow trout (35% in common carp) crude protein by “Pearsons’s Square”

(Figure A 3.1).

31

Table A3.2 Target composition of test diets (% of DM).

Rainbow trout Common carp Crude protein 42 35 Crude fat 15 10

Figure A3.1 Hand formulation of balanced diet

Example: Hand formulation of balanced diet for common carp

32

Compare for each commodity the estimated inclusion rate with the maximum

recommended inclusion rate presented in the feedstuff table (Annex 2).

If the estimated inclusion rate of a commodity is by more than 5% higher than the

recommended inclusion rate, select the ingredient from the same group (PC or CC) with

the second highest residue level and recalculate the formulation using the average protein

content of both ingredients (assuming 50:50% mixing).

Example:

Corn meal – the estimated inclusion rate (31.7%) is below the maximum recommended inclusion

rate (35%) for common carp.

Peanut meal – the estimated inclusion rate (68.3%) is higher than the maximum recommended

inclusion rate (35% for common carp. Peanut meal should be mixed with the protein concentrate

having the second highest STMR-P (soybean meal).

Peanut / soybean mix (50:50)

(46.5%CP+49.8%CP)/2 = 48.15%CP

recalculate the balanced diet

Example: Hand formulation of balanced diet for common carp with protein concentrate mix

33

Calculate the fat content of the formulated diet containing protein concentrate (PC) and

carbohydrate concentrate (CC) based on the lipid content of the individual ingredients

(Annex 2).

Example:

The feed formulation contains:

32.7% peanut meal (65.4/2) containing 1% crude lipids

32.7% soybean meal (65.4/2) containing 0.8% crude lipids

34.6% corn meal containing 4.8% crude lipids

The formulated feed has a lipid content of 2.25%

Subtract the estimated lipid content from the target lipid content (15% Rainbow trout or

10% Common carp) defined for the test species to calculate the required fat supplement.

Adjust the fat content of the diet by addition of the plant derived oil with the highest

STMR to reach a dietary lipid content of 15% Rainbow trout (10% Common carp).

The inclusion of fat (vegetable oil) will reduce the amount of CC and PC in the feed

formulation. Therefore, the mixture of both ingredients has to be adjusted to maintain the

target protein concentration of 42% (Rainbow trout) or 35% (Common carp). A 5% fat

inclusion for instance will leave 95% of the diet to cover the protein requirement. The

target protein needs to be increased accordingly to:

TargetNew (%CP) = Targetoriginal(%CP)*(100/95)

Recalculate the amount of PC and CC according to Figure A3.1.

34

Example:

10% (Target lipid content) – 2.25% (lipid content of PC and CC) = 7.75% (required fat

supplement)

The 7.75% fat inclusion will leave 92.25% of the diet to cover the protein requirement. The target

protein needs to be increased accordingly:

35(%CP)*(100/92.25) = 37.9% (new target protein)

The formulated feed contains:

33.67% peanut meal (92.25%/100*73%/2) containing 1% crude lipids

33.67% soybean meal (92.25%/100*73%/2) containing 0.8% crude lipids

24.91% corn meal (92.25%/100*27%) containing 4.8% crude lipids

7.75% plant oil

The formulated feed has a lipid content of 9.6%1)

Calculate the dietary burden of the formulated diet (mg/kg) based on the STMR-P values

of all ingredients.

35

Example

Final formulation (%)

STMR-P (mg/kg DM)

Dietary burden (mg/kg DM)

Peanut meal (PC) 33.67 0.09 0.03 Soybean meal (PC) 33.67 0.05 0.017 Corn meal (CC) 24.91 0.3 0.075 Vegetable oil (F) 7.75 0.01 0.001

Σ 0.123

1) The lipid content of the formulated diet should not deviate by more than 1% and 1.5% from the target lipid content for Common carp (10%) and Rainbow trout (15%), respectively. Otherwise the formulation should be optimized by linear programming.