Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance)

Recommended species	Recommended range of test temperature (°C)	Recommended total length of test animal (cm) [^E0117]
Danio rerio [^E0116] (Teleostei, Cyprinidae) (Hamilton-Buchanan) Zebra-fish	20 – 25	3,0 ± 0,5
Pimephales promelas (Teleostei, Cyprinidae) (Rafinesque) Fathead minnow	20 – 25	5,0 ± 2,0
Cyprinus carpio (Teleostei, Cyprinidae) (Linnaeus) Common carp	20 – 25	8,0 ± 4,0 [^E0118]
Oryzias latipes (Teleostei, Poecilliidae) (Temminck and Schlegel) Ricefish	20 – 25	4,0 ± 1,0
Poecilia reticulata (Teleostei, Poeciliidae) (Peters) Guppy	20 – 25	3,0 ± 1,0
Lepomis macrochirus (Teleostei Centrarchidae) (Rafinesque) Bluegill	20 – 25	5,0 ± 2,0
Oncorhynchus mykiss (Teleostei Salmonidae) (Walbaum) Rainbow trout	13 – 17	8,0 ± 4,0
Gasterosteus aculeatus (Teleostei, (Gasterosteidae) (Linnaeus) Three-spined stickleback	18 — 20	3,0 ± 1,0

Meyer et al. (1)

It should be noted that in the test itself weight is preferred as the measure for size and growth rate constant derivations. It is however recognised that length is a more practical measure if fish have to be selected by sight at the beginning of an experiment (i.e. from the stock population).

This length range is indicated in the Testing Methods for New Chemical Substances etc. based on the Japan's Chemical Substances Control Law (CSCL).

Various estuarine and marine species have less widely been used, for example:

Spot	(Leiostomus xanthurus)
Sheepshead minnow	(Cyprinodon variegatus)
Silverside	(Menidia beryllina)
Shiner perch	(Cymatogaster aggregata)
English sole	(Parophrys vetulus)
Staghorn sculpin	(Leptocottus armatus)
Three-spined stickleback	(Gasterosteus aculeatus)
Sea bass	(Dicentracus labrax)
Bleak	(Alburnus alburnus)

The freshwater fish listed in the table above are easy to rear and/or are widely available throughout the year, whereas the availability of marine and estuarine species is partially confined to the respective countries. They are capable of being bred and cultivated either in fish farms or in the laboratory, under disease- and parasite-controlled conditions, so that the test animal will be healthy and of known parentage. These fish are available in many parts of the world.

LITERATURE:

• (1)Meyer A., Biermann C.H. and Orti G. (1993), The phylogenetic position of the zebrafish (Danio rerio), a model system in developmental biology: An invitation to the comparative method Proc. R. Soc. Lond. B. 252: 231-236.

Appendix 4

SAMPLING SCHEDULES FOR AQUEOUS AND DIETARY EXPOSURE TESTS

1. Theoretical example of a sampling schedule for a full aqueous exposure bioconcentration test of a substance with log KOW = 4.

Fish Sampling	Sample time schedule	Sample time schedule	No. of water samples [^E0119]	No. of fish per sample [^E0119]
Fish Sampling	Minimal required frequency (days) [^E0120]	Additional sampling (days) [^E0120]	No. of water samples [^E0119]	No. of fish per sample [^E0119]
Uptake phase
1	– 1		2 [^E0121]	4 [^E0122]
	0		(2)	(3 [^E0124])
2	0,3		2	4
		0,4	(2)	(4)
3	0,6		2	4
		0,9	(2)	(4)
4	1,2		2	4
		1,7	(2)	(4)
5	2,4		2	4
		3,3	(2)	(4)
6	4,7		2	4 – 8 [^E0123]
				(3 [^E0124])
Depuration phase				Transfer fish to water free of test substance
7	5,0		2	4
		5,3		(4)
8	5,9		2	4
		7,0		(4)
9	9,3		2	4
		11,2		(4)
10	14,0		2	4 – 8 [^E0123]
		17,5		(4+3 [^E0124])
TOTAL				40 – 72 (48 – 80) [^E0123]

Values in brackets are numbers of samples (water, fish) to be taken if additional sampling is carried out.

Pre-test estimate of k 2 for log K OW of 4,0 is 0,652 days^{– 1}. The total duration of the experiment is set to 3 × t SS = 3 × 4,6 days, i.e. 14 days. For the estimation of t SS refer to Appendix 5.

Sample water after a minimum of 3 ‘chamber-volumes’ has been delivered.

These fish are sampled from the stock population.

If greater precision or metabolism studies are necessary that require more fish, these should be sampled particularly at the end of the uptake and depuration phases (cf. paragraph 40).

At least 3 additional fish may be required for lipid content analysis if it is not possible to use the same fish sampled for substance concentrations at the start of the test, the end of the uptake phase and the end of the depuration phase. Note it should be possible in many cases to use the 3 control fish alone (cf. paragraph 56).

2. Theoretical example of sampling schedule for dietary bioaccumulation test of substance following 10 day uptake and 42 day depuration phases.

Sampling event	Sample time schedule	Sample time schedule	No. food samples	No. fish per sample	No. fish per sample
Sampling event	Day of phase	Additional fish samples?	No. food samples	Test Group	Control Group
Uptake phase
1	0	Possible [^E0125] [^E0126]	3 — test group	0	5 – 10
1	0	Possible [^E0125] [^E0126]	3 — control group [^E0125]		(8 – 13) [^E0126]
1A [^E0127]	1-3			5 – 10	5 – 10
2	10	Yes [^E0128]	3 — test group	10 – 15 [^E0128]	5 – 10
2	10	Yes [^E0128]	3 — control group [^E0125]	(13 – 18) [^E0129]	(8 – 13) [^E0129]
Depuration phase
3	1	Yes [^E0128]		10 – 15 [^E0128]	5 – 10
4	2			5 – 10	5 – 10
5	4			5 – 10	5 – 10
6	7	Yes [^E0128]		10 – 15 [^E0128]	5 – 10
7	14			5 – 10	5 – 10
8	28			5 – 10	5 – 10
9	42	Yes [^E0128]		10 – 15 [^E0128] (13 – 18) [^E0129]	5 – 10 (8 – 13) [^E0129]
TOTAL				59 – 120 (63 – 126) [^E0128] [^E0129]	50 – 110 (56 – 116) [^E0128] [^E0129]

3 samples of feed from both control and test groups analysed for test substance concentrations and for lipid content.

Fish are sampled from the stock population as near to the start of the study as possible; at least 3 fish from the stock population at test start should be sampled for lipid content.

(Optional) sampling early in the uptake phase provides data to calculate dietary assimilation of test substance that can be compared with the assimilation efficiency calculated from the depuration phase data.

5 extra fish may be sampled for tissue-specific analysis.

At least 3 additional fish may be required for lipid content analysis if it is not possible to use the same fish sampled for substance concentrations at the start of the test, the end of the uptake phase and the end of the depuration phase. Note it should be possible in many cases to use the 3 control fish alone (cf. paragraphs 56 and 153).

Note on phase and sampling timings: The uptake phase begins with the first feeding of spiked diet. An experimental day runs from one feeding until shortly before the next, 24 hours later. The first sampling event (1 in the table) should be taken shortly before the first feeding (e.g. one hour). Sampling during a study should ideally be carried out shortly before the following day's feeding (i.e. about 23 hours after the sample day's feeding). The uptake phase ends shortly before the first feeding with unspiked diet, when the depuration phase begins (test group fish are likely to be still digesting spiked feed in the intervening 24 hours after the last spiked diet feeding). This means that the end of uptake sample should be taken shortly before the first feeding with unspiked diet and the first depuration phase sample should be taken about 23 hours after the first feeding with unspiked feed.

Appendix 5

GENERAL CALCULATIONS

• 1.Introduction

• 2.Prediction of the duration of the uptake phase

• 3.Prediction of the duration of the depuration phase

• 4.Sequential method: determination of depuration (loss) rate constant k2

• 5.Sequential method: determination of uptake rate constant k1 (aqueous exposure method only)

• 6.Simultaneous method for calculation of uptake and depuration (loss) rate constants (aqueous exposure method only)

• 7.Growth dilution correction for kinetic BCF and BMF

• 8.Lipid normalisation to 5 % lipid content (aqueous exposure method only)

1. INTRODUCTION

The general fish aquatic bioaccumulation model can be described in terms of uptake and loss processes, ignoring uptake with food. The differential equation (dC f/dt) describing the rate of change in fish concentration (mg·kg^{– 1}·day^{– 1}) is given by (1):

Image omitted; see official source	[Equation A5.1]

Where

k 1	=	First order rate constant for uptake into fish (l·kg– 1·day– 1).
---	---	---
k 2	=	First order rate constant for depuration from fish (day– 1).
---	---	---
kg	=	First order rate constant for fish growth (‘growth dilution’) (day– 1)
---	---	---
km	=	First order rate constant for metabolic transformation (day– 1)
---	---	---
ke	=	First order rate constant for faecal egestion (day– 1)
---	---	---
C w	=	Concentration in water (mg·l– 1).
---	---	---
Cf	=	Concentration in fish (mg·kg– 1 wet weight).

For bioaccumulating substances, it can be expected that a time-weighted average (TWA) is the most relevant exposure concentration in water (Cw) within the allowed range of fluctuation (cf. paragraph 24). It is recommended to calculate a TWA water concentration, according to the procedure in Appendix 6 of TM C.20 (2). It should be noted that the ln-transformation of the water concentration is suitable when exponential decay between renewal periods is expected, e.g. in a semi-static test design. In a flow through system, ln-transformation of exposure concentrations may not be needed. If TWA water concentrations are derived, they should be reported and used in subsequent calculations.

In a standard fish BCF test uptake and depuration can be described in terms of two first order kinetic processes.

Rate of uptake = k 1 × C w	[Equation A5.2]
Overall loss rate = (k 2 + k g + k m + k e) × C f	[Equation A5.3]

At steady-state, assuming growth and metabolism are negligible (i.e. the values for k g and k m cannot be distinguished from zero), the rate of uptake equals the rate of depuration, and so combining Equation A5.2 and Equation A5.3 gives the following relationship:

Image omitted; see official source	[Equation A5.4]

Where

C f-SS	=	Concentration in fish at steady-state (mg kg– 1 wet weight).
---	---	---
C w-SS	=	Concentration in water at steady-state (mg l– 1).

The ratio of k 1/k 2 is known as the kinetic BCF (BCFK) and should be equal to the steady-state BCF (BCFSS) obtained from the ratio of the steady-state concentration in fish to that in water, but deviations may occur if steady-state was uncertain or if corrections for growth have been applied to the kinetic BCF. However, as k 1 and k 2 are constants, steady-state does not need to be reached to derive a BCFK.

Based on these first order equations, this Appendix 5 includes the general calculations necessary for both aqueous and dietary exposure bioaccumulation methods. However, sections 5, 6 and 8 are only relevant for the aqueous exposure method but are included here as they are ‘general’ techniques. The sequential (sections 4 and 5) and simultaneous (section 6) methods allow the calculation of uptake and depuration constants which are used to derive kinetic BCFs. The sequential method for determining k 2 (section 4) is important for the dietary method as it is needed to calculate both assimilation efficiency and BMF. Appendix 7 details the calculations that are specific to the dietary method.

2. PREDICTION OF THE DURATION OF THE UPTAKE PHASE

Before performing the test, an estimate of k 2 and hence some percentage of the time needed to reach steady-state may be obtained from empirical relationships between k 2 and the n-octanol/water partition coefficient (K OW) or k 1 and BCF. It should be realised, however, that the equations in this section only apply when uptake and depuration follow first-order kinetics. If this is clearly not the case it is advised to seek advice from a biostatistician and/or pharmacokineticist, if predictions of the uptake phase are desirable.

An estimate of k 2 (day^{– 1}) may be obtained by several methods. For example, the following empirical relationships could be used in the first instance ([^E0130]):

log k 2 = 1,47 – 0,414logKOW	(r2=0,95) [(3); Equation A5.5]

or

Image omitted; see official source	[Equation A5.6]
Where k 1 = 520 × W – 0,32 (for substances with a log K OW > 3)	(r 2=0,85)[(4); Equation A5.7]
And Image omitted; see official source	(r 2=0,90)[(5); Equation A5.8]

W = mean treated fish weight (grams wet weight) at the end of uptake/start of depuration ([^E0131])

For other related relationships see (6). It may be advantageous to employ more complicated models in the estimation of k2 if, for example, it is likely that significant metabolism may occur (7) (8). However as the complexity of the model increases, greater care should be taken with the interpretation of the predictions. For example the presence of nitro groups might indicate fast metabolism, but this is not always the case. Therefore the user should weigh up the predictive method results against chemical structure and any other relevant information (for example preliminary studies) in the scheduling of a study.

The time to reach a certain percentage of steady-state may be obtained, by applying the k 2-estimate, from the general kinetic equation describing uptake and depuration (first-order kinetics), assuming growth and metabolism is negligible. If substantial growth occurs during the study, the estimations described below will not be reliable. In such cases, it is better to use the growth corrected k 2g as described later (see Section 7 of this Appendix):

Image omitted; see official source	[Equation A5.9]

or, if C w is constant:

Image omitted; see official source	[Equation A5.10]

When steady-state is approached (t → ∞), Equation A5.10 may be reduced (cf. (9) (10)) to:

Image omitted; see official source	[Equation A5.11]

or

Image omitted; see official source	[Equation A5.12]

Then BCF × C w is an approximation to the concentration in the fish at steady-state (C f-SS). [Note: the same approach can be used when estimating a steady-state BMF with the dietary test. In this case, BCF is replaced with BMF and C w with C food, concentration in the food, in the equations above]

Equation A5.10 may be transcribed to:

Image omitted; see official source	[Equation A5.13]

or

Image omitted; see official source	[Equation A5.14]

Applying Equation A5.14, the time to reach a certain percentage of steady-state may be predicted when k 2 is pre-estimated using Equation A5.5 or Equation A5.6.

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 of the concentration of the test substance in fish plotted against linear time to reach at least 50 % of steady-state (i.e. 0,69/k 2), but not more than 95 % of steady-state (i.e. 3,0/k 2) (11). In case accumulation reaches beyond 95 % of steady-state, calculation of a BCFSS becomes feasible.

The time to reach 80 percent of steady-state is (using Equation A5.14):

Image omitted; see official source	[Equation A5.15]

or

Image omitted; see official source	[Equation A5.16]

Similarly the time to reach 95 percent of steady-state is:

Image omitted; see official source	[Equation A5.17]

For example, the duration of the uptake phase (i.e. time to reach a certain percentage of steady-state, e.g. t 80 or t 95) for a test substance with log K OW = 4 would be (using Equation A5.5, Equation A5.16 and Equation A5.17):

logk 2 = 1,47 – 0,414 · 4

k 2 = 0,652 day^{– 1}

Image omitted; see official source

or

Image omitted; see official source

Alternatively, the expression:

teSS = 6,54 · 10 – 3 · K OW + 55,31 (hours)	[Equation A5.18]

may be used to calculate the time for effective steady-state (teSS) to be reached (12). For a test substance with log K OW = 4 this results in:

teSS = 6,54 · 10 ^{– 3} · 10⁴ + 55,31 = 121 hours

3. PREDICTION OF THE DURATION OF THE DEPURATION PHASE

A prediction of the time needed to reduce the body burden to a certain percentage of the initial concentration may also be obtained from the general equation describing uptake and depuration (assuming first order kinetics, cf. Equation A5.9 (1) (13).

For the depuration phase, C w (or C food for the dietary test) is assumed to be zero. The equation may then be reduced to:

Image omitted; see official source	[Equation A5.19]

or

Image omitted; see official source	[Equation A5.20]

where C f,0 is the concentration at the start of the depuration period.

50 percent depuration will then be reached at the time (t 50):

Image omitted; see official source

or

Image omitted; see official source

Similarly 95 percent depuration will be reached at:

Image omitted; see official source

If 80 % uptake is used for the first period (1,6/k 2) and 95 % loss in the depuration phase (3,0/k 2), then depuration phase is approximately twice the duration of the uptake phase.

Note that the estimations are based on the assumption that uptake and depuration patterns will follow first order kinetics. If first-order kinetics is obviously not obeyed, these estimations are not valid.

4. SEQUENTIAL METHOD: DETERMINATION OF DEPURATION (LOSS) RATE CONSTANT K

2**

Most bioconcentration data have been assumed to be ‘reasonably’ well described by a simple two-compartment/two-parameter model, as indicated by the rectilinear curve which approximates to the points for concentrations in fish (on an ln scale), during the depuration phase.

Image omitted; see official source

Text of image

In(concentration)

Cf2

Cf1

t2

t1

time

Note that deviations from a straight line may indicate a more complex depuration pattern than first order kinetics. The graphical method may be applied for resolving types of depuration deviating from first order kinetics.

To calculate k 2 for multiple time (sampling) points, perform a linear regression of ln(concentration) versus time. The slope of the regression line is an estimate of the depuration rate constant k 2 ([^E0132]). From the intercept the average concentration in the fish at the start of the depuration phase (C 0,d; which equals the average concentration in the fish at the end of the uptake phase) can easily be calculated (including error margins) (59) :

C 0, d = eintercept	[Equation A5.21]

To calculate k 2 when only two time (sampling) points are available (as in the minimised design), substitute the two average concentrations into the following equation

Image omitted; see official source	[Equation A5.22]

Where ln(C f1) and ln(C f2) are the natural logarithms of the concentrations at times t 1 and t 2, respectively, and t 2 and t 1 are the times when the two samples were collected relative to the start of depuration ([^E0133]).

5. SEQUENTIAL METHOD: DETERMINATION OF UPTAKE RATE CONSTANT K

1 (AQUEOUS EXPOSURE METHOD ONLY)**

To find a value for k1 given a set of sequential time concentration data for the uptake phase, use a computer program to fit the following model:

Image omitted; see official source	[Equation A5.23]

Where k 2 is given by the previous calculation, C f(t) and C w(t) are the concentrations in fish and water, respectively, at time t.

To calculate k 1 when only two time (sampling) points are available (as in the minimised design), use the following formula:

Image omitted; see official source	[Equation A5.24]

Where k 2 is given by the previous calculation, C f is the concentration in fish at the start of the depuration phase, and C w is the average concentration in the water during the uptake phase ([^E0134]).

Visual inspection of the k 1 and k 2 slopes when plotted against the measured sample point data can be used to assess goodness of fit. If it turns out that the sequential method has given a poor estimate for k 1 then the simultaneous approach to calculate k 1 and k 2 should be applied (see next section 6). Again, the resulting slopes should be compared against the plotted measured data for visual inspection of goodness of fit. If the goodness of fit is still poor this may be an indication that first order kinetics do not apply and other more complex models should be employed.

6. SIMULTANEOUS METHOD FOR CALCULATION OF UPTAKE AND DEPURATION (LOSS) RATE CONSTANTS (AQUEOUS EXPOSURE METHOD ONLY)

Computer programs can be used to find values for k 1 and k 2 given a set of sequential time concentration data and the model:

Image omitted; see official source	0 < t < t c	[Equation A5.25]
Image omitted; see official source	t > t c	[Equation A5.26]

where

t c	=	time at the end of the uptake phase.

This approach directly provides standard errors for the estimates of k 1 and k

1/k 2 is substituted by BCF (cf. Equation A5.4) in Equation A5.25 and Equation A5.26, the standard error and 95 % CI of the BCF can be estimated as well. This is especially useful when comparing different estimates due to data transformation. The dependent variable (fish concentration) can be fitted with or without ln transformation, and the resulting BCF uncertainty can be evaluated.

As a strong correlation exists between the two parameters k 1 and k 2 if estimated simultaneously, it may be advisable first to calculate k 2 from the depuration data only (see above); k 2 in most cases can be estimated from the depuration curve with relatively high precision. k 1 can be subsequently calculated from the uptake data using non-linear regression ([^E0135]). It is advised to use the same data transformation when fitting sequentially.

Visual inspection of the resulting slopes when plotted against the measured sample point data can be used to assess goodness of fit. If it turns out that this method has given a poor estimate for k1 then the simultaneous approach to calculate k 1 and k 2 can be applied. Again, the fitted model should be compared against the plotted measured data for visual inspection of goodness of fit and the resulting parameter estimates for k 1, k 2 and resulting BCF and their standard errors and/or confidence intervals should be compared between different types of fit.

If the goodness of fit is poor this may be an indication that first order kinetics does not apply and other more complex models should be employed. One of the most common complications is fish growth during the test.

7. GROWTH DILUTION CORRECTION FOR KINETIC BCF AND BMF

This section describes a standard method for correction due to fish growth during the test (so called ‘growth dilution’) which is only valid when first order kinetics applies. In case there are indications that first order kinetics do not apply, it is advised to seek advice from a biostatistician for a proper correction of growth dilution or to use the mass based approach described below.

In some cases this method for correcting growth dilution is subject to a lack of precision or sometimes does not work (for example for very slowly depurating substances tested in fast growing fish the derived depuration rate constant corrected for growth dilution, k 2g, may be very small and so the error in the two rate constants used to derive it become critical, and in some cases k g estimates may be larger than k 2). In such cases an alternative approach (i.e. mass approach), which also works when first order growth kinetics have not been obeyed, can be used which avoids the need for the correction. This approach is outlined at the end of this section.

Growth rate constant subtraction method for growth correction

For the standard method all individual weight and length data are converted to natural logarithms and ln(weight) or ln(1/weight) is plotted vs. time (day), separately for treatment and control groups. The same process is carried out for the data from the uptake and depuration phases separately. Generally for growth dilution correction it is more appropriate to use the weight data from the whole study to derive the growth rate constant (k g), but statistically significant differences between the growth rate constants derived for the uptake phase and depuration phase may indicate that the depuration phase rate constant should be used. Overall growth rates from aqueous studies for test and control groups can be used to check for any treatment related effects.

A linear least squares correlation is calculated for the ln(fish weight) vs. day (and for ln(1/weight) vs. day) for each group (test(s) and control groups, individual data, not daily mean values) for the whole study, uptake and depuration phases using standard statistical procedures. The variances in the slopes of the lines are calculated and used to evaluate the statistical significance (p = 0,05) of the difference in the slopes (growth rate constants) using the student t-test (or ANOVA if more than one concentration is tested). Weight data are generally preferred for growth correction purposes. Length data, treated in the same way, may be useful to compare control and test groups for treatment related effects. If there is no statistically significant difference in the weight data analysis, the test and control data may be pooled and an overall fish growth rate constant for the study (k

The calculated growth rate constant (k g expressed as day^-1) can be subtracted from the overall depuration rate constant (k 2) to give the depuration rate constant, k 2g.

k 2 g = k 2 – kg	[Equation A5.27]

The uptake rate constant is divided by the growth-corrected depuration rate constant to give the growth-corrected kinetic BCF, denoted BCFKg (or BMFKg).

Image omitted; see official source	[Equation A5.28]

The growth rate constant derived for a dietary study is used in Equation A7.5 to calculate the growth corrected BMFKg (cf. Appendix 7).

Mass based method for growth correction

An alternative to the above ‘growth rate constant subtraction method’ that avoids the need to correct for growth can be used as follows. The principle is to use depuration data on a mass basis per whole fish rather than on a concentration basis.

Convert depuration phase tissue concentrations (mass of test substance/unit mass of fish) into mass of test substance/fish: match concentrations and individual fish weights in tabular form (e.g. using a computer spreadsheet) and multiply each concentration by the total fish weight for that measurement to give a set of mass test substance/fish for all depuration phase samples.

Plot the resulting natural logarithm of substance mass data against time for the experiment (depuration phase) as would be done normally.

For the aqueous exposure method, derive the uptake rate constant routinely (see sections 4 and 6) note that the ‘normal’ k 2 value should be used in the curve fitting equations for k 1) and derive the depuration rate constant from the above data. Because the resulting value for the depuration rate constant is independent of growth as it has been derived on a mass basis per whole fish, it should be denoted as k 2g and not k 2.

8. LIPID NORMALISATION TO 5 % LIPID CONTENT (AQUEOUS EXPOSURE METHOD ONLY)

BCF results (kinetic and steady-state) from aqueous exposure tests should also be reported relative to a default fish lipid content of 5 % wet weight, unless it can be argued that the test substance does not primarily accumulate in lipid (e.g. some perfluorinated substances may bind to proteins). Fish concentration data, or the BCF, need to be converted to a 5 % lipid content wet weight basis. If the same fish were used for measuring substance concentrations and lipid contents at all sampling points, this requires each individual measured concentration in the fish to be corrected for that fish's lipid content.

Image omitted; see official source	[Equation A5.29]

where

C f,L	=	lipid-normalised concentration in fish (mg kg– 1 wet weight)
---	---	---
L	=	lipid fraction (based on wet weight)
---	---	---
C f	=	concentration of test substance in fish (mg kg– 1 wet weight)

If lipid analysis was not conducted on all sampled fish, a mean lipid value is used to normalise the BCF. For the steady-state BCF, the mean value recorded at the end of the uptake phase in the treatment group should be used. For the normalisation of a kinetic BCF there may be some cases where a different approach is warranted, for example if the lipid content changed markedly during the uptake or depuration phase. However a feeding rate that minimises dramatic changes in lipid content should be used anyway routinely.

Image omitted; see official source	[Equation A5.30]

where

BCFKL	=	lipid-normalised kinetic BCF (L kg– 1)
---	---	---
L n	=	mean lipid fraction (based on wet weight)
---	---	---
BCFK	=	kinetic BCF (L kg– 1)

LITERATURE:

• (1) Arnot J.A. and Gobas F.A.P.C. (2004), A food web bioaccumulation model for organic chemicals in aquatic ecosystems, Environ. Toxicol. Chem. 23: 2343–2355.

• (2) Chapter C.20 of this Annex, Daphnia magna Reproduction Test.

• (3) Spacie A. and Hamelink J.L. (1982), Alternative models for describing the bioconcentration of organics in fish. Environ. Toxicol. Chem. 1: 309-320.

• (4) Sijm D.T.H.M., Verberne M.E., de Jonge W.J., Pärt P. and Opperhuizen A. (1995), Allometry in the uptake of hydrophobic chemicals determined in vivo and in isolated perfused gills. Toxicol. Appl. Pharmacol. 131: 130-135.

• (5) Bintein S., Devillers J. and Karcher W. (1993), Nonlinear dependence of fish bioconcentration on n-octanol/water partition coefficient. SAR QSAR Environ. Res. 1: 29-39.

• (6) Kristensen P. (1991), Bioconcentration in fish: comparison of BCF's derived from OECD and ASTM testing methods; influence of particulate matter to the bioavailability of chemicals. Danish Water Quality Institute, Hørsholm, Denmark.

• (7) Arnot J.A., Meylan W., Tunkel J., Howard P.H., Mackay D., Bonnell M. and Boethling R.S. (2009), A quantitative structure-activity relationship for predicting metabolic biotransformation rates for organic chemicals in fish. Environ. Toxicol. Chem. 28: 1168-1177.

• (8) OECD (2011), QSAR Toolbox 2.1. February 2011. Available from: http://www.oecd.org/document/54/0,3746,en_2649_34379_42923638_1_1_1_1,00.html.

• (9) Branson D.R., Blau G.E., Alexander H.C. and Neely W.B. (1975). Bioconcentration of 2,2′,4,4′ tetrachlorobiphenyl in rainbow trout as measured by an accelerated test. T. Am. Fish. Soc. 104: 785-792.

• (10) Ernst W. (1985), Accumulation in aquatic organisms, in Appraisal of tests to predict the environmental behaviour of chemicals, Sheeman, P., et al., Editors. John Wiley & Sons Ltd, New York, NY, USA: 243-255.

• (11) Reilly P.M., Bajramovic R., Blau G.E., Branson D.R. and Sauerhoff M.W. (1977), Guidelines for the optimal design of experiments to estimate parameters in first order kinetic models. Can. J. Chem. Eng. 55: 614-622.

• (12) Hawker D.W. and Connell D.W. (1988), Influence of partition coefficient of lipophilic compounds on bioconcentration kinetics with fish. Wat. Res. 22: 701-707.

• (13) Konemann H. and van Leeuwen K. (1980), Toxicokinetics in fish: Accumulation and elimination of six chlorobenzenes by guppies. Chemosphere. 9: 3-19.

Appendix 6

EQUATION SECTION FOR AQUEOUS EXPOSURE TEST: MINIMISED TEST DESIGN

The rationale for this approach is that the bioconcentration factor in a full test can either be determined as a steady-state bioconcentration factor (BCFSS) by calculating the ratio of the concentration of the test substance in the fish's tissue to the concentration of the test substance in the water, or by calculating the kinetic bioconcentration factor (BCFK) as the ratio of the uptake rate constant k 1 to the depuration rate constant k

If a measurement of the concentration of the substance in tissues (C f1) is made at the time that exposure ends (t 1) and the concentration in tissue (C f2) is measured again after a period of time has elapsed (t 2), the depuration rate constant (k 2) can be estimated using Equation A5.22 from Appendix 5.

The uptake rate constant, k 1, can then be determined algebraically using Equation A5.23 from Appendix 5 (where C f equals C f1 and t equals t 1) (1). The kinetic bioconcentration factor for the minimised design (designated as BCFKm to distinguish it from kinetic bioconcentration factors determined using other methods) is thus:

Image omitted; see official source	[Equation A6.1]

Concentrations or results should be corrected for growth dilution and normalised to a fish lipid content of 5 %, as is described in Appendix 5.

The minimised BCFSS is the BCF calculated at the end of the uptake phase, assuming that steady-state has been reached. This can only be assumed, as the number of sampling points is not sufficient for proving this.

Image omitted; see official source	[Equation A6.2]

Where

C f-minSS = Concentration in fish at assumed steady-state at end of uptake (mg kg^{– 1} wet weight).

C w-minSS = Concentration in water at assumed steady-state at end of uptake (mg l^{– 1}).

LITERATURE:

• (1)Springer T.A., Guiney P.D., Krueger H.O. and Jaber M.J. (2008), Assessment of an approach to estimating aquatic bioconcentration factors using reduced sampling. Environ. Toxicol. Chem. 27: 2271-2280.

Appendix 7

EQUATION SECTION FOR DIETARY EXPOSURE TEST

• 1.Example of constituent quantities of a suitable commercial fish food

• 2.Food spiking technique examples

• 3.Calculation of assimilation efficiency and biomagnification factor

• 4.Lipid correction

• 5.Evaluation of differences between measured time zero concentration (C0,m) and derived time zero concentration (C0,d)

• 6.Guidance for very fast depurating test substances

1. EXAMPLE OF CONSTITUENT QUANTITIES OF A SUITABLE COMMERCIAL FISH FOOD

Major constituent	Fish meal
Crude Protein	≤ 55,0 %
Crude fat	≤ 15,0 % [^E0136]
Crude Fibre	≥ 2,0 %
Moisture	≥ 12 %
Ash	≥ 8 %

In some regions it may only be possible to obtain fish food with a lipid concentration that falls far short of this upper limit. In such cases studies should be run with the lower lipid concentration in the food as supplied, and the feeding rate adjusted appropriately to maintain fish health. Diet lipids should not be artificially increased by the addition of excess oil.

2. FOOD SPIKING TECHNIQUE EXAMPLES

General Points

Control diets should be prepared in exactly the same way as the spiked diet, but with an absence of test substance.

To check the concentration of the treated diet, triplicate samples of the dosed food should be extracted with a suitable extraction method and the test substance concentration or radioactivity in the extracts measured. High analytical recoveries (> 85 %) with low variation between samples (three sample concentrations for the substance taken at test start should not vary more than ± 15 % from the mean) should be demonstrated.

During the dietary test, three diet samples for analysis should be collected on day 0 and at the end of the uptake phase for the determination of the test substance content in the diet.

Fish food preparation with a liquid test material (neat)

A target, nominal test concentration in the treated fish food is set, for example 500 μg test substance/g food. The appropriate quantity (by molar mass or specific radioactivity) of neat test substance is added to a known mass of fish food in a glass jar or rotary evaporator bulb. The mass of fish food should be sufficient for the duration of the uptake phase (taking into account the need for increasing quantities at each feed owing to fish growth). The fish feed/test substance should be mixed overnight by slow tumbling (e.g. using a roto-rack mixer or by rotation if a rotary evaporator bulb is used). The spiked diet should be stored under conditions that maintain stability of the test substance within the feed mix (e.g. refrigeration) until use.

Fish food preparation with a corn or fish oil vehicle

Solid test substances should be ground in a mortar to a fine powder. Liquid test substances can be added directly to the corn or fish oil. The test substance is dissolved in a known quantity of corn or fish oil (e.g. 5-15 ml). The dosed oil is quantitatively transferred into a rotary evaporation bulb of suitable size. The flask used to prepare the dosed oil should be flushed with two small aliquots of oil and these added to the bulb to make sure all dissolved test substance is transferred. To ensure complete dissolution/dispersion in the oil (or if more than one test substance is being used in the study), a micro-stirrer is added, the flask stoppered and the mixture stirred rapidly overnight. An appropriate quantity of fish diet (usually in pellet form) for the test is added to the bulb, and the bulb's contents are mixed homogeneously by continuously turning the glass bulb for at least 30 minutes, but preferably overnight. Thereafter, the spiked food is stored appropriately (e.g. refrigerated) to ensure test substance stability in the food until use.

Fish food preparation with an organic solvent

An appropriate quantity of test substance (by molar mass or specific radioactivity) sufficient to achieve the target dose is dissolved in a suitable organic solvent (e.g. cyclohexane or acetone; 10-40 ml, but a greater volume if necessary depending on the quantity of food to spike). Either an aliquot, or all (added portion wise), of this solution is mixed with the appropriate mass of fish food sufficient for the test to achieve the required nominal dose level. The food/test substance can be mixed in a stainless steel mixing bowl and the freshly-dosed fish food left in the bowl in a laboratory hood for two days (stirred occasionally) to allow the excess solvent to evaporate, or mixed in a rotary evaporator bulb with continuous rotation. The excess solvent can be ‘blown’ off under a stream of air or nitrogen if necessary. Care should be taken to ensure that the test substance does not crystallise as the solvent is removed. The spiked diet should be stored under conditions (e.g. refrigeration) that maintain stability of the test substance within the feed mix until use.

3. CALCULATION OF ASSIMILATION EFFICIENCY AND BIOMAGNIFICATION FACTOR

To calculate the assimilation efficiency, the overall depuration rate constant should first be estimated according to section 4 of Appendix 5 (using the ‘sequential method’, i.e. standard linear regression) using mean sample concentrations from the depuration phase. The feeding rate constant, I, and uptake duration, t, are known parameters of the study. C food, the mean measured concentration of the test substance in the food is a measured variable in the study. C 0,d, the test substance concentration in the fish at the end of the uptake phase, is usually derived from the intercept of a plot of ln(concentration) vs. depuration day.

The substance assimilation efficiency (a, absorption of test substance across the gut) is calculated as:

Image omitted; see official source	[Equation A7.1]

where:

C 0,d = derived concentration in fish at time zero of the depuration phase (mg kg^{– 1});

k 2 = overall (not growth-corrected) depuration rate constant (day^{– 1}), calculated according to equations in Appendix 5, Section 3;

I = food ingestion rate constant (g food g^{– 1} fish day^{– 1});

C food = concentration in food (mg kg^{– 1} food);

t = duration of the feeding period (day)

However, the feeding rate, I, used in the calculation may need to be adjusted for fish growth to give an accurate assimilation efficiency, a. In a test where fish grow significantly during the uptake phase (in which no correction of feed quantities is made to maintain the set feeding rate), the effective feeding rate as the uptake phase progresses will be lower than that set, resulting in a higher 'real' assimilation efficiency. (Note this is not important for the overall calculation of BMF as the I terms effectively cancel out between Equation A7.1 and Equation A7.4). The mean feeding rate corrected for growth dilution, I g, can be derived in several ways, but a straightforward and rigorous one is to use the known growth rate constant (k

Image omitted; see official source	[Equation A7.2]

where

W f(t) = mean fish weight at uptake day t

W f,0 = mean fish weight at the start of the experiment

In this way (at least) the mean fish weight on the last day of exposure (W f,end-of-uptake) can be estimated. As the feeding rate was set based on W f,0, the effective feeding rate for each day of uptake can be calculated using these two weight values. The growth-corrected feeding rate, I g (g food g^-1 fish day^{– 1}), to use instead of I in cases of rapid growth during the uptake phase, can then be calculated as

Image omitted; see official source	[Equation A7.3]

Once the assimilation efficiency has been obtained, the BMF can be calculated by multiplying it with the feeding rate constant I (or I g, if used to calculate α) and dividing the product by the overall depuration rate constant k 2:

Image omitted; see official source	[Equation A7.4]

The growth-corrected biomagnification factor should also be calculated in the same way, using the growth corrected depuration rate constant (as derived according to section 7 in Appendix 5). Again, if I g has been used to calculate α, it should also be used here instead of I:

Image omitted; see official source	[Equation A7.5]

where:

α = assimilation efficiency (absorption of test substance across the gut);

k 2 = overall (not growth-corrected) depuration rate constant (day^{– 1}), calculated according to equations in Appendix 5, Section 3;

k 2g = growth-corrected depuration rate constant (day^{– 1});

I = food ingestion rate constant (g food g^{– 1} fish day^{– 1});

The growth-corrected half-life (t 1/2) is calculated as follows.

Image omitted; see official source	[Equation A7.6]

The substance assimilation efficiency from the diet can also be estimated if tissue residues are determined during the linear phase of the uptake phase (between days 1 and 3). In this case the substance assimilation efficiency (α) can be determined as follows

Image omitted; see official source	[Equation A7.7]

Where

C fish (t) = the concentration of test substance in the fish at time t (mg kg^{– 1} wet weight).

4. LIPID CORRECTION

If lipid content was measured on the same fish as chemical analysis for all sampling intervals, then individual concentrations should be corrected on a lipid basis and the ln(concentration, lipid corrected) plotted against depuration (day) to give C 0,d and k

food on a lipid basis (i.e. C food is multiplied by the mean lipid fraction of the food). Subsequent calculation using Equation A7.4 and Equation A7.5 will give the lipid-corrected (and growth-dilution corrected) BMF directly.

Otherwise, the mean lipid fraction (w/w) in the fish and in the food are derived for both treatment and control groups (for food and control group fish this is usually from data measured at exposure start and end; for treatment group fish this is usually from data measured at end of exposure only). In some studies, fish lipid content may increase markedly; in such cases it is more appropriate to use a mean test fish lipid concentration calculated from the measured values at the end of exposure and end of depuration. In general, data from the treatment group only should be used to derive both of the lipid fractions.

The lipid-correction factor (Lc) is calculated as:

Image omitted; see official source	[Equation A7.8]

where L fish and L food are the mean lipid fractions in fish and food, respectively.

The lipid-correction factor is used to calculate the lipid-corrected biomagnification factor (BMFL):

Image omitted; see official source	[Equation A7.9]

5. EVALUATION OF DIFFERENCES BETWEEN MEASURED TIME ZERO CONCENTRATION (C

0,M) AND DERIVED TIME ZERO CONCENTRATION (C 0,D)**

The measured time zero concentration (C 0,m) and derived time zero concentration (C 0,d) should be compared. If they are very similar, then this supports the first order model used to derive the depuration parameters.

In some studies there may be a marked difference between the derived time zero value, C 0,d, and the mean measured time zero concentration. C 0,m (see last bullet point of paragraph 159 of this test method). If C 0,d is very much lower than C 0,m (C 0,d << C 0,m), the difference may suggest the presence of undigested spiked food in the gut. This may be tested experimentally by conducting separate analysis on the excised gut if additional (whole fish) samples were taken and stored at the end of the uptake phase. Otherwise, if a statistically valid outlier test applied to the depuration phase linear regression indicates that the first sample point of depuration is erroneously elevated, carrying out the linear regression to derive k 2 but omitting the first depuration concentration point may be appropriate. In such cases, if the uncertainty in the linear regression is greatly decreased, and it is clear that approximately first order depuration kinetics were obeyed, it may be appropriate to use the resulting C 0,d and k 2 values in the assimilation efficiency calculation. This should be fully justified in the report. It is also possible that non-first order kinetics were operating in the depuration phase. If this is likely (i.e. the natural logarithm transformed data appear to follow a curve compared with the straight-line linear regression plot), then the calculations of k 2 and C 0,d are unlikely to be valid and the advice of a biostatician should be sought.

If C 0,d is very much higher than the measured value (C 0,d >> C 0,m) this may indicate: that the substance was depurated very fast (i.e. sampling points approached the limit of quantification of the analytical method very early in the depuration phase, cf. Section 6 below); that there was a deviation from first order depuration kinetics; that the linear regression to derive k 2 and C 0,d is flawed; or that a problem with the measured concentrations in the study occurred at some sampling time points. In such cases the linear regression plot should be scrutinised for evidence of samples at or near the limit of quantification, for outliers and for obvious curvature (suggestive of non-first order kinetics), and highlighted in the report. Any subsequent re-evaluation of the linear regression to improve estimated values should be described and justified. If marked deviation from first order kinetics is observed, then the calculations of k 2 and C 0,d are unlikely to be valid and the advice of a biostatician should be sought.

6. GUIDANCE FOR VERY FAST DEPURATING TEST SUBSTANCES

As discussed in paragraph 129 of the test method, some substances may depurate so fast that a reliable time zero concentration, C 0,d, and k 2 cannot be derived because in samples very early in the depuration phase (i.e. from the second depuration sample onwards) the substance is effectively no longer measured (concentrations reported at the limit of quantification). This situation was observed in the ring test carried out in support of this test method with benzo[a]pyrene, and has been documented in the validation report for the method. In such cases linear regression cannot be carried out reliably, and is likely to give an unrealistically high estimate of C 0,d, resulting in an apparent assimilation efficiency much greater than 1. It is possible to calculate a conservative estimate of k 2 and an ‘upper bound’ BMF in these instances.

Using those data points of the depuration phase where a concentration was measured, up to and including the first ‘non-detect’ concentration (concentration set at limit of quantification), a linear regression (using natural logarithm transformed concentration data against time) will give an estimate of k

2 can be estimated using Equation A5.22 in Appendix 5. This k2 estimate can be used to estimate an assimilation efficiency according to equation A7.1, substituting the C0,d value in the equation with the measured time zero concentration (C0,m) in cases where C0,d is clearly estimated to be much higher than could have been achievable in the test. If C0,m was not measureable, then the limit of detection in fish tissue should be used. If, in some cases, this gives a value of α > 1, then the assimilation efficiency is assumed to 1 as a ‘worst case’.

The maximum BMFK can then be estimated using Equation A7.4, and should be quoted as a ‘much less than’ (<<) value. For example, for a study carried out with a feeding rate of 3 % and a depuration half-life less than 3 days, and a ‘worst case’ α of 1, the BMFK is likely to be below about 0,13. Given the purpose of this estimation and the fact that values will be conservative in nature, it is not necessary to correct them for growth dilution or fish and food lipid content.

Appendix 8

APPROACHES TO ESTIMATE TENTATIVE BCFS FROM DATA COLLECTED IN THE DIETARY EXPOSURE STUDY

The dietary method is included in this test method for the bioaccumulation testing of substances that cannot in practice be tested using the aqueous exposure method. The aqueous exposure method gives a bioconcentration factor, whereas the dietary method leads directly to information on feeding biomagnification potential. In many chemical safety regimes information on aquatic bioconcentration is required (for example in risk assessment and the Globally Harmonization System of Classification). Hence there is a need to use the data generated in a dietary study to estimate a bioconcentration factor that is comparable to tests conducted according to the aqueous exposure method ([^E0137]). This section explores approaches that may be followed to do this, while recognising the shortcomings that are inherent in the estimations.

The dietary study measures depuration to give a depuration rate constant, k

The estimation of an uptake rate constant for water exposure of a test substance is reliant on many assumptions, all of which will contribute to the estimate's uncertainty. Furthermore, this approach to estimating a BCF assumes that the overall rate of depuration (including contributory factors like distribution in the body and individual depuration processes) is independent of the exposure technique used to produce a test substance body burden.

The main assumptions inherent in the estimation approach can be summarised as follows.

Depuration following dietary uptake is the same as depuration following aqueous exposure for a given substance

Uptake from water would follow first order kinetics

Depending on the method used to estimate uptake:

• — uptake can be correlated with fish weight alone

• — uptake can be correlated with the substance's octanol-water partition coefficient alone

• — uptake can be correlated with a combination of fish weight and the substance's octanol-water partition coefficient

• — factors that can affect uptake in an aqueous exposure study in practice such as substance bioavailability, adsorption to apparatus, molecular size etc. have little effect

• — and, crucially:

The database (‘training set’) used to develop the uptake estimation method is representative of the substance under consideration

Several publications in the open literature have derived equations relating uptake from water in fish via the gills to a substance's octanol-water partition coefficient, fish weight (1) (2) (3) (4), volume and/or lipid content, membrane permeation/diffusion (5) (6), fish ventilation volume (7) and by a fugacity/mass balance approach (8) (9) (10). A detailed appraisal of such methods in this context is given in Crookes & Brooke (11). A publication by Barber (12) focussed on modelling bioaccumulation through dietary uptake is also useful in this context as it includes contributions from gill uptake rate models. A section of the background document to the 2004 dietary protocol (13) was also devoted to this aspect.

Most of these models seem to have been derived using limited databases. For models where details of the database used to build the model are available, it appears that the types of substances used are often of a similar structure or class (in terms of functionality, e.g. organochlorines). This adds to the uncertainty in using a model to predict an uptake rate constant for a different type of substance, in addition to test-specific considerations like species, temperature, etc.

A review of available techniques (11) highlighted that no one method is ‘more correct’ than the others. Therefore, a clear justification should be given for the model used. Where several methods are available for which the use can be justified, it may be prudent to present several estimates of k 1 (and so BCF) or a range of k 1 values (and BCF) according to several uptake estimation methods. However, given the differences in model types and datasets used to develop them, taking a mean value from estimates derived in different ways would not be appropriate.

Some researchers have postulated that BCF estimates of this sort require a bioavailability correction to account for a substance's adsorption to dissolved organic carbon (DOC) under aqueous exposure conditions, to bring the estimate in line with results from aqueous exposure studies (e.g. (13) (14)). Howeverl this correction may not be appropriate given the low levels of DOC required in an aqueous exposure study for a ‘worst case’ estimate (i.e. ratio of bioavailable substance to substance as measured in solution). For highly hydrophobic substances uptake at the gill may become limited by the rate of passive diffusion near the gill surface; in this case it is possible that the correction may be accounting for this effect rather than what it was designed for.

It is advised to focus on methods that require inputs for which data will be readily available for substances tested according to the dietary study described here (i.e. log K OW, fish weight). Other methods that require more complex inputs may be applied, but may need additional measurements in the test or detailed knowledge on the test substance or fish species that may not be widely available. In addition, choice of model may be influenced by the level of validation and applicability domain (see (11) for a review and comparison of different methods).

It should be borne in mind that the resulting k 1 estimate, and estimated BCF, are uncertain and may need to be treated in a weight-of-evidence approach along with the derived BMF and substance parameters (e.g. molecular size) for an overall picture of a substance's bioaccumulation potential. Interpretation and use of these parameters may depend on the regulatory framework.

LITERATURE:

• (1) Sijm D.T.H.M., Pärt P. and Opperhuizen A. (1993), The influence of temperature on the uptake rate constants of hydrophobic compounds determined by the isolated perfused gills of rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 25: 1-14.

• (2) Sijm D.T.H.M., Verberne M.E., Part P. and Opperhuizen A. (1994), Experimentally determined blood and water flow limitations for uptake of hydrophobic compounds using perfused gills of rainbow trout (Oncorhynchus mykiss): Allometric applications. Aquat. Toxicol. 30: 325-341.

• (3) Sijm D.T.H.M., Verberne M.E., de Jonge W.J., Pärt P. and Opperhuizen A. (1995), Allometry in the uptake of hydrophobic chemicals determined in vivo and in isolated perfused gills. Toxicol. Appl. Pharmacol. 131: 130-135.

• (4) Barber M.C. (2003), A review and comparison of models for predicting dynamic chemical bioconcentration in fish. Environ. Toxicol. Chem. 22: 1963-1992.

• (5) Opperhuizen A. (1986), Bioconcentration of hydrophobic chemicals in fish, in Aquatic Toxicology and Environmental Fate, STP 921, Poston, T.M. and Purdy, R., Editors. American Society for Testing and Materials, Philadelphia, PA, USA: 304-315.

• (6) Arnot J.A. and Gobas F.A.P.C. (2004), A food web bioaccumulation model for organic chemicals in aquatic ecosystems. Environ. Toxicol. Chem. 23: 2343-2355.

• (7) Thomann R.V. (1989), Bioaccumulation model of organic chemical distribution in aquatic food chains. Environ. Sci. Technol. 23: 699-707.

• (8) Hendriks A.J., van der Linde A., Cornelissen G. and Sijm D.T.H.M. (2001). The power of size. 1. Rate constants and equilibrium ratios for accumulation of organic substances related to octanol-water partition ratio and species weight. Environ. Toxicol. Chem. 20: 1399-1420.

• (9) Campfens J. and Mackay D. (1997), Fugacity-based model of PCB bioaccumulation in complex aquatic food webs. Environ. Sci. Technol. 31: 577-583.

• (10) Arnot J.A. and Gobas F.A.P.C. (2003), A generic QSAR for assessing the bioaccumulation potential of organic chemicals in aquatic food webs. QSAR Comb. Sci. 22: 337-345.

• (11) Crookes M. and Brooke D. (2010), Estimation of fish bioconcentration factor (BCF) from depuration data. Draft Report. Environmental Agency, Bristol, UK.

• (12) Barber M.C. (2008), Dietary uptake models used for modelling the bioaccumulation of organic contaminants in fish. Environ. Toxicol. Chem. 27: 755-777

• (13) Anonymous (2004), Background document to the fish dietary study protocol, document submitted to the TC-NES WG on PBT.

• (14) Gobas F. and Morrison H. (2000), Bioconcentration and biomagnification in the aquatic environment, in Handbook of property estimation methods for chemicals, Boethling, R.S. and Mackay, D., Editors. Lewis Publishers, Boca Racton, FL, USA: 189-231.

C.14.   FISH JUVENILE GROWTH TEST

1. METHOD

This growth toxicity test method is a replicate of the OECD TG 215 (2000).

1.1.   INTRODUCTION

This test is designed to assess the effects of prolonged exposure to chemicals on the growth of juvenile fish. It is based on a method, developed and ring-tested (1)(2) within the European Union, for assessing the effects of chemicals on the growth of juvenile rainbow trout (Oncorynchus mykiss) under flow-through conditions. Other well documented species may be used. For example, experience has been gained from growth tests with zebrafish (Danio rerio) ([^E0138]) (3)(4) and ricefish (medaka, Oryzias latipes) (5)(6)(7).

See also General introduction Part C.

1.2.   DEFINITIONS

Lowest observed effect concentration (LOEC): is the lowest tested concentration of a test substance at which the substance is observed to have a significant effect (at p < 0,05 ) when compared with the control. However, all test concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC.

No observed effect concentration (NOEC): is the test concentration immediately below the LOEC.

EC x: in this test method is the concentration of the test substance which causes a x % variation in growth rate of the fish when compared with controls.

Loading rate: is the wet weight of fish per volume of water.

Stocking density: is the number of fish per volume of water.

Individual fish specific growth rate: expresses the growth rate of one individual based on its initial weight.

Tank-average specific growth rate: expresses the mean growth rate of a tank population at one concentration.

Pseudo specific growth rate: expresses the individual growth rate compared to the mean initial weight of the tank population.

1.3.   PRINCIPLE OF THE TEST METHOD

Juvenile fish in exponential growth phase are placed, after being weighted, in test chambers and are exposed to a range of sublethal concentrations of the test substance dissolved in water preferably under flow-through, or, if not possible, under appropriate semi-static (static-renewal) conditions. The test duration is 28 days. Fish are fed daily. The food ration is based on initial fish weights and may be recalculated after 14 days. At the end of the test, the fish are weighed again. Effects on growth rates are analysed using a regression model in order to estimate the concentration that would cause a x % variation in growth rate, i.e. ECx (e.g. EC10, EC20, or EC30). Alternatively, the data may be compared with control values in order to determine the lowest observed effect concentration (LOEC) and hence the no observed effect concentration (NOEC).

1.4.   INFORMATION ON THE TEST SUBSTANCE

Results of an acute toxicity test (see Test Method C. 1.) preferably performed with the species chosen for this test, should be available. This implies that the water solubility and the vapour pressure of the test substance are known and a reliable analytical method is available for the quantification of the substance in the test solutions with known and reported accuracy and limit of detection is available.

Useful information includes the structural formula, purity of the substance, stability in water and light, pKa, Pow and results of a test for ready biodegradability (see Test Method C.4).

1.5.   VALIDITY OF THE TEST

For the test to be valid the following conditions apply:

• — the mortality in the control(s) must not exceed 10 % at the end of the test;

• — the mean weight of fish in the control(s) must have increased enough to permit the detection of the minimum variation of growth rate considered as significant. A ring-test (2) has shown that for rainbow trout the mean weight of fish in the controls must have increased by at least the half (i.e. 50 %) of their mean initial weight over 28 days; e.g. initial weight: 1 g/fish (= 100 %), final weight after 28 days: ≥ 1,5 g/fish (≥ 150 %);

• — the dissolved oxygen concentration must have been at least 60 % of the air saturation value (ASV) throughout the test;

• — the water temperature must not differ by more than ± 1 ^oC between test chambers at any one time during the test and should be maintained within a range of 2 ^oC within the temperature ranges specified for the test species (Appendix 1).

1.6.   DESCRIPTION OF THE TEST METHOD

1.6.1.   Apparatus

Normal laboratory equipment and especially the following:

• — oxygen and pH meters;

• — equipment for determination of water hardness and alkalinity;

• — adequate apparatus for temperature control and preferably continuous monitoring;

• — tanks made of chemically inert material and of suitable capacity in relation to the recommended loading and stocking density (see Section 1.8.5 and Appendix 1);

• — suitably accurate balance (i.e. accurate to ± 0,5 %).

1.6.2.   Water