N-ethyl-N-[2-[1-(2-methylpropoxy)ethoxy]ethyl]-4-(phenylazo)aniline

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15/12/2017 to 08/02/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study, conducted to GLP and current methodology.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Radiolabelling:

no

Details on sampling:

Collection of diet samples.
Samples of the treatment, reference, and control diets were collected and analysed in triplicate for the test item and reference substances at the beginning and end of the uptake phase.
The total lipid content of the prepared diet was sampled in triplicate prior to the start of the test and at the end of the uptake phase.

Collection of fish samples.
At each sampling interval, a sufficient number of fish were collected to provide 6 replicates samples from the control, treatment and reference group. Fish were impartially removed from the test chambers, and euthanized. Fish were euthanized in order to carry out the measurements of weight and length and chemical analysis. For this, fish were placed for about five minutes in a beaker containing MS222 (CAS number 886-86-2) at a lethal concentration of approximately 300 mg.L-1. On day 0 and 14 of uptake phase and at the end of depuration phase, additional fish were collected to determine lipid content.

Vehicle:

yes

Remarks:

corn oil

Details on preparation of test solutions, spiked fish food or sediment:

Fish were fed with Small Granular product supplied by Special Diet Services (www.sdsdiets.fr).
Test and reference items were dissolved in corn oil as vehicle before mixing with fish food. The amount of corn oil (ref: ACRO405435000 ; VWR distributor) added to the fish food was 0.5%. The same amount of corn oil was added to the control diet.

Test organisms (species):

Pimephales promelas

Details on test organisms:

The fathead minnow, Pimephales promelas, was used in the study. This species was obtained from Osage Catfisheries (1170 Nichols Road, Osage Beach, MO 65065 , USA). All fish were on the same year class and come from the same source. Identification of the species was verified by the supplier. Fish arrived in the lab on 25/08/2017.

Route of exposure:

feed

Justification for method:

dietary exposure method used because stable, measurable water concentrations cannot be maintained

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

14 d

Total depuration duration:

28 d

Hardness:

6.5 °dH

Test temperature:

22.1 °C

pH:

7.1

Dissolved oxygen:

7.20%

TOC:

0.2505 - 0.7926 (mg.L-1)

Salinity:

Not specified

Conductivity:

253.4 (μS/cm)

Details on test conditions:

Test conditions
A continuous-flow was used to deliver water to the treatment, reference and control groups. The flow of water to the test chambers was controlled by peristaltic pumps
The delivery of water by peristaltic pumps was checked prior to the test and at approximately weekly intervals thereafter.
The test chambers used during both uptake and depuration phases were 96-L glass aquaria and filled with approximately 78 L of water.

Water
The water used for holding and testing was freshwater obtained from a mixture of deionized water and tap water issued from our water system.

Preliminary trial
A non-GLP pilot study was conducted to evaluate the biomagnification potential of test item. The pilot study was conducted with different nominal concentration of test item in diet.
The procedures used to prepare the fortified test diet for the definitive study were established during the pilot study. The stability, homogeneity and storage conditions of the test substance in the treated diet were confirmed during the pilot study. The results of the pilot study showed that there were not any harmful effect on fish at 500μg/ Yellow 124 and there was uptake of test item into the fish tissue, BMF values were estimated to be 0.00008.

Preparation of diet
Fish were fed with Small Granular product supplied by Special Diet Services (www.sdsdiets.fr).
Test and reference items were dissolved in corn oil as vehicle before mixing with fish food. The amount of corn oil (ref: ACRO405435000 ; VWR distributor) added to the fish food was 0.5%. The same amount of corn oil was added to the control diet. The preparation of the different diets is described below under "any other information on materials and methods inc tables".

Feeding of fish
During the uptake phase, the fish were fed with the appropriate test item, reference substance, or control diet daily at approximately 1.5 % of body weight (wet weight). The initial feed rate was based on weight measurements of a sample of fish collected on first day of acclimation and on first day of the test.
The amount of feed was adjusted based on the wet weights of sampled fish at each interval during testing period to account for growth during the test. Any uneaten food as well feces were removed from the different test chambers shortly after feeding.

Acclimation and test initiation
An acclimation period was performed from December 15th , 2017.
Fathead minnows were collected from the rearing tanks and impartially distributed, using dip nets, to the different test chambers until they contained 80 fish.
Initiation of the test was performed on December 27th, 2017, corresponding to the day 0 of the uptake phase.

Fish-to-water loading rate
Loading rate was defined as the total wet weight of fish per liter of water that passed through the test chamber in 24 hours.
On first day of acclimation, 10 fish were sampled in each test chamber and then weighed to estimate the feed rate.
Loading rates at the start of the acclimation were 0.44, 0.45 and 0.44 g.L-1.day-1 for the control, the test item and the reference item group, respectively.
On first day of uptake, 10 fish were sampled in each test chamber and then weighed to estimate the feed rate. 9 out 10 were used to chemical analysis and lipid content, one was replaced in the test corresponding chamber.
Loading rates at the start of the test were 0.40, 0.40 and 0.41 g.L-1.day-1 for the control, the test item and the reference item group, respectively.
Loading rate during the test is details in annex X and ranged from 0.22 to 0.43 g.L-1.day-1 in the control group, from 0.22 to 0.43 g.L-1.day-1 in the test item group and from 0.22 to 0.41 g.L-1.day-1 in the reference item group.

Biological observations
All fish were observed daily to evaluate the number or mortalities and the number of individuals exhibiting signs of abnormal behavior.

Environmental conditions
Neon day light was used for illumination of the controlled rooms. A photoperiod of 16 hours of light and 8 hours of dark was applied. Light intensity at the surface of the water at the start of the uptake phase was measured using a light meter.
The target water test temperature during the test was 22 ± 2°C. Temperature was measured in all test chambers at the beginning and end of the test and at approximately weekly intervals during the test. Temperature was also monitored continuously only in the control test chamberà.
Measurements of Dissolved oxygen, pH, conductivity, were performed in each test chamber at the beginning and at the end of the test and approximately weekly during the test using a multiparemeter. Hardness and alkalinity was measured in the control test chambers at the beginning and end of the uptake phase and at the end of the depuration phase.

Nominal and measured concentrations:

Nominal: Control: 500 μg CI Yellow 124. g-1 fish food; 10 μg HCB. g-1 fish food (reference)
Mean Measured: <0.02 μg/g; 374.0 μg CI Yellow 124. g-1 fish food; 8.4 μg HCB. g-1 fish food

Reference substance (positive control):

yes

Remarks:

Hexachlorobenzene

Details on estimation of bioconcentration:

The depuration (loss) rate constant (k2)is the numerical value defining the rate of reduction in the concentration of the test substance in the test fish (or specified tissues thereof) following the transfer of the test fish from a medium containing the test substance to a medium free of that substance (k2 is expressed in day-1).
Dissolved organic carbon (DOC): measure of the concentration of carbon originating from dissolved organic sources in the test media.
Exposure or uptake phase: time during which the fish are exposed to the test chemical.
Food ingestion rate (I): Average amount of food eaten by each fish each day, relative to the estimated average fish whole body weight (expressed in terms of g food-1.g fish.day-1).
Kinetic bioconcentration factor (BCFK): ratio of the uptake rate constant, k1, to the depuration rate constant, k2 (i.e. k1/ k2 – see corresponding definitions in this annex). In principle the value should be comparable to the BCFSS (see definition above), but deviations may occur if steadystate was uncertain or if corrections for growth have been applied to the kinetic BCF.
The lipid normalised kinetic bioconcentration factor (BCFKL) is normalised to a fish with a 5% lipid content.
The lipid normalised, growth corrected kinetic bioconcentration factor (BCFKgL) is normalised to a fish with a 5% lipid content and corrected for growth during the study period as described in Annex 5.The lipid normalised steady-state bioconcentration factor (BCFSSL) is normalised to a fish with 5% lipid content.
The octanol-water partition coefficient (KOW) is the ratio of a chemical’s solubility in n-octanol and water at equilibrium (OECD Guidelines 107, 117, 123); also expressed as POW. The logarithm of KOW is used as an indication of a chemical’s potential for bioconcentration by aquatic organisms.
A steady-state is reached in the plot of test substance in fish (Cf) against time when the curve becomes parallel to the time axis and three successive analyses of Cf made on samples taken at intervals of at least two days are within ± 20% of each other, and there is no significant increase of Cf in time between the first and last successive analysis. When pooled samples are analysed at least four successive analyses are required. For test substances which are taken up slowly the intervals would more appropriately be seven days.
The steady-state bioconcentration factor (BCFSS) does not change significantly over a prolonged period of time, the concentration of the test substance in the surrounding medium being constant during this period of time (cf. Definition of steady-state).
The uptake rate constant (k1) is the numerical value defining the rate of increase in the concentration of test substance in/on test fish (or specified tissues thereof) when the fish are exposed to that chemical (k1) is expressed in L. Kg-1.day-1).

Lipid content:

8.7 %

Time point:

start of exposure

Lipid content:

10.3 %

Time point:

end of exposure

Key result

Conc. / dose:

374 µg/g food

Temp.:

>= 20 - <= 24 °C

pH:

7

Type:

BMF

Value:

0.001 dimensionless

Basis:

normalised lipid fraction

Time of plateau:

15 d

Calculation basis:

steady state

Key result

Conc. / dose:

374 µg/g food

Temp.:

>= 20 - <= 24 °C

pH:

7

Type:

BCF

Value:

43.3 L/kg

Basis:

whole body w.w.

Calculation basis:

other: OECD calculation matrix

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

3.5 d

Key result

Rate constant:

overall depuration rate constant (d-1)

Value:

-0.206

Details on kinetic parameters:

Depuration rate and Derived time Zero
The depuration rate constant (k2) was calculated by plotting the natural logarithm of measured concentrations of the test or reference substance (ln(concentration)) for the depuration period versus time (day). A linear least squares correlation was calculated for the ln(concentration) vs. time (day) data. The slope of the line was reported as the overall depuration constant (k2) and the intercept of the line was reported as the natural logarithm of the derived time zero concentration (C0,d). The overall depuration rate constants (k2) and the derived time zero concentrations (C0,d) for the treatment reference groups are presented.
In the test item treatment the k2 value for whole fish tissue was -0.2064. The derived time zero concentration (C0,d) for whole fish tissues in the test item treatment group was 3.14 μg/g.

Assimilation efficiency
The assimilation efficiency (α) is the efficiency of absorption of the test or reference substance across the gut.
The assimilation efficiency for whole fish tissue in the test item treatment group was 0.0068.

Lipid correction
The corrections were calculated using (i) the mean lipid fractions from the fish (Lfish) from Day 0 and 14 of uptake and from day 28 of depuration and (ii) The mean lipid fractions of diet (Lfood) analyzed on Day 0 and 14 of uptake.
The lipid-correction factor was calculated to be 0.53 for the treatment group.

Metabolites:

Not determined.

Results with reference substance (positive control):

Concentration of reference item in fish tissue
The mean measured tissue concentration of test item in whole fish tissues at Day 14 of uptake was 1,06 μg/g. The whole fish tissue concentration from Day 14 of the uptake phase (1,06 μg/g ) is also the measured time zero concentration (C0,m). The mean measured concentration of NOMSUBSTANCE in whole fish tissue by Day 28 of depuration was 0.38 μg/g.

The growth and lipid corrected BMF value for the reference substance HCB was 0,4553 in whole fish tissues. The time to reach 95% steady state (BMFSS) was 90 days and the estimated time to reach 50% clearance was 18 days.

Depuration rate and Derived time Zero
In the HCB reference group, the k2 value for whole fish tissue was -0.0387. The derived time zero concentration (C0,d) for whole fish tissues in the HCB reference group was 1.18 μg/g.

The assimilation efficiency for whole fish tissue in the HCB reference group was 0.5278.

Lipid correction
The corrections were calculated using (i) the mean lipid fractions from the fish (Lfish) from Day 0 and 14 of uptake and from day 28 of depuration and (ii) The mean lipid fractions of diet (Lfood) analyzed on Day 0 and 14 of uptake.
The lipid-correction factor was calculated to be 0.52 for the reference group.

Details on results:

Observations and clinical signs of toxicity
There were no observed mortalities in reference groups during the tests. There was one mortality in the control group on Day 2 of the uptake phase. There was one mortality in the test item group on Day 8 of the depuration phase. This was incidental and not considered treatment related. All fish appeared normal and healthy throughout the test.

Concentration of test item in fish tissues
The mean measured tissue concentration of test item in whole fish tissues at Day 14 of uptake was 7.1 μg/g. The whole fish tissue concentration from Day 14 of the uptake phase (7,1 μg/g) is also the measured time zero concentration (C0,m). The mean measured concentration of test item in whole fish tissue by Day 28 of depuration was 0.04 μg/g
The concentrations of test item in the control group was < 0.02 μg/g during the test.

Growth rate
Growth measurements (length and total wet weight) for individual fish collected during uptake and depuration in each condition are presented in appendix.
Growth rate constants (kg), were calculated by performing a linear least squares correlation on the individual data of the control, treatment and reference groups.
The slopes of the linear regression were compared statistically using the student’s t-test (p = 0.05).
In each condition, there were no statistical differences between the slopes of the uptake and depuration phase growth data in the treatment group (p > 0.05) therefore the uptake and depuration data was pooled.
The growth rate constants (kg) were -0.0093, -0.0067 and -0.0052 day-1 in the control, treatment and reference groups, respectively.

Food ingestion rate
The food ingestion rate constant (I) was calculated using the desired amount of food to be fed per day, weight of fish and number of fish remaining in each tank. Feeding rates were adjusted based out on the weights of sampled fish at each interval during the study. The food ingestion rate was calculated to be 0.015 g food g-1 fish day-1 (i.e. 1,5%).

BCF estimation based on BMF values
According to the Guidance Document OECD TG 305, different methods are available to estimate the BCF from a dietary exposure. For this, the Excel BCF Estimation Tool version 2 available on the OECD website was used (http://www.oecd.org/chemicalsafety/testing/section-3-environmental-fate-behaviour-software-tg-305.htm).

With this tool, estimated BCF ranged from 43.3 to 4054 according to the method.
In this study, This BMFKgL estimate is reasonable (0.001), as the measured assimilation efficiency approaches zero, indicating that the test substance is not taken up from the food and the depuration rate is not really relevant.
Measured concentrations of the test substance in the feed were 374,0 µg Y124 /g , whereas maximum concentration in tissues at end of uptake was 0.182 µg Y124 /g. This would seem to indicate that test item simply does not pass through the gut lining into the tissues, and suggests that there is a large margin between the observed accumulation of the test substance and any level of accumulation that might be of concern.
Thus several lines of evidence indicate the empirical method of Inoue et.al. (2012) should be favoured, and the best available estimate of BCFKgL is 43.3.

Please see "any other information on results incl tables" below for further iformation.

Physico-chemical measurements in each test chamber are summarized in the table below:

		Dissolved oxygen (%)	pH	Temperature (°C)	Conductivity (µS/cm)	Lightning (lux)	Hardness (°dH)	Alkalinity (mg /L CaCO3)
Control	Mean	75.8	7.1	22.1	257.7	891.5	6.1	108.5
	Standard deviation	8.0	0.2	0.2	22.1	2.1	1.3	23.9
	RSD (%)	10.6	2.2	0.7	8.6	0.2	22.0	22.0
	Min	64.6	6.9	21.4	228.2	890.0	4.6	82.8
	Max	85.8	7.3	22.5	278.9	893.0	7.3	130.1
Treatment_Y124	Mean	72.0	7.1	22.1	253.4	890.0	6.5	116.6
	Standard deviation	6.0	0.2	0.3	19.3	46.0	1.1	20.5
	RSD (%)	8.3	2.5	1.4	7.6	5.2	17.6	17.6
	Min	64.2	7.0	21.8	228.9	925.0	5.2	93.5
	Max	81.3	7.5	22.7	270.4	990.0	7.4	132.6
Reference_HCB	Mean	73.4	7.1	21.9	247.2	940.0	6.4	113.9
	Standard deviation	3.9	0.2	0.3	21.5	19.8	1.8	32.7
	RSD (%)	5.4	2.4	1.4	8.7	2.1	28.7	28.7
	Min	67.4	7.0	21.9	225.5	863.0	4.3	76.2
	Max	77.6	7.5	22.7	272.9	891.0	7.5	134.6

The dissolved oxygen concentration was > 60% of the air saturation value throughout the test in each test chamber. The pH was in all conditions within a range of ± 0.5. Water did not differ by more than 2°C in treatment or control groups.

 

Concentration of lipid in control, test and reference diets

Lipids (%)
Phase_time (days)		Control	Treatment_Y124	Reference_HCB
Uptake_0		14,9	13,8	14,1
		13,9	14,1	14,4
		13,8	14,2	14,1
	mean	14,2	14,0	14,2
	SD	0,6	0,2	0,2
Uptake_14		14,6	14,5	14,1
		14,5	14,7	14,5
		14,3	14,7	14,4
	mean	14,4	14,6	14,3
	SD	0,1	0,1	0,2
	mean	14,3	14,3	14,3
	SD	0,4	0,4	0,2

 

Concentration of test item in test and control diets

The concentration of test item in the test diet and control diet collected at day 0 and 14 of the uptake phase is presented in table below.

[Y124] µg.g food-1
Phase_time (days)		Control	Treatment_Y124
Uptake_0		<0,02	369,0
		<0,02	364,5
		<0,02	374,3
	mean	NA	369,3
	mean +15%	NA	424,6
	mean -15%	NA	313,9
	SD	NA	4,9
Uptake_14		<0,02	378,2
		<0,02	382,6
		<0,02	375,7
	mean	NA	378,8
	mean +15%	NA	435,6
	mean -15%	NA	322,0
	SD	NA	3,5
	General mean	NA	374,0
	General mean +20%	NA	448,8
	General mean -20%	NA	299,2

 

Test item was not detected in control diet

The mean measured concentration of the test item in diet was 374,0 µg.g food^-1. The concentration of the test item in fish diet before and at the end of the uptake phase was within ± 20% .

The homogeneity of the test item in fish diet did not vary more than ± 15% from the mean.

 

Concentration of reference item in reference diet

[HCB] µg.g food-1
Phase_time (days)		Treatment_Y124
Uptake_0		8,40
		9,10
		9,02
	mean	8,8
	mean +15%	10,2
	mean -15%	7,5
	SD	0,4
Uptake_14		7,73
		7,62
		7,45
	mean	7,6
	mean +15%	8,7
	mean -15%	6,5
	SD	0,1
	General mean	8,2
	General mean +20%	9,9
	General mean -20%	6,6

The mean measured concentration of the reference item in diet was 8,2 µg.g food^-1.

The concentration of the reference item in fish diet before and at the end of the uptake phase was within ± 20% .

The homogeneity of the reference item in fish diet did not vary more than ± 15% from the mean.

 

Concentration of test item in fish tissues

The concentrations of test items in tissues of fish exposed to the treatment diet with a mean measured concentration of 374.0 µg/g are summarized in the table below .

Sampling date	Phase_time (days)	Mean	SD
27/12/2017	Uptake_0	<0.02	<0.02
10/01/2018	Uptake_14	7.10	3.34
11/01/2018	Depuration_1	3.07	2.04
12/01/2018	Depuration_2	4.44	4.26
18/01/2018	Depuration_5	3.04	2.55
18/01/2018	Depuration_8	0.67	0.84
25/01/2018	Depuration_15	0.18	0.21
01/02/2018	Depuration_22	0.07	0.11
07/02/2018	Depuration_28	0.04	0.07

 

The mean measured tissue concentration of test item in whole fish tissues at Day 14 of uptake was 7.1 µg/g. The whole fish tissue concentration from Day 14 of the uptake phase (7,1 µg/g) is also the measured time zero concentration (C_0,m). The mean measured concentration of test item in whole fish tissue by Day 28 of depuration was 0.04 µg/g

 

Concentration of reference item in fish tissue

The concentrations of test items in tissues of fish exposed to the reference diet with a mean measured concentration of 8.2 µg/g are presented in appendix X and summarized in the table below:

Sampling date	Phase_time (days)	Mean (µg/g)	SD
27/12/2017	Uptake_0	0,03	0,01
10/01/2018	Uptake_14	1,06	0,36
11/01/2018	Depuration_1	1,09	0,28
12/01/2018	Depuration_2	1,07	0,30
18/01/2018	Depuration_5	1,19	0,29
18/01/2018	Depuration_8	0,87	0,25
25/01/2018	Depuration_15	0,74	0,33
01/02/2018	Depuration_22	0,61	0,19
07/02/2018	Depuration_28	0,38	0,16

The mean measured tissue concentration of test item in whole fish tissues at Day 14 of uptake was 1,06 µg/g. The whole fish tissue concentration from Day 14 of the uptake phase (1,06 µg/g ) is also the measured time zero concentration (C_0,m). The mean measured concentration of test item in whole fish tissue by Day 28 of depuration was 0.38 µg/g.

 

Depuration rate and Derived time Zero

The depuration rate constant (k₂) was calculated by plotting the natural logarithm of measured concentrations of the test or reference substance (ln(concentration)) for the depuration period versus time (day). A linear least squares correlation was calculated for the ln(concentration)vs.time (day) data. The slope of the line was reported as the overall depuration constant (k₂) and the intercept of the line was reported as the natural logarithm of the derived time zero concentration (C_0,d). The overall depuration rate constants (k₂) and the derived time zero concentrations (C_0,d) for the treatment reference groups are presented.

In the test item treatment thek₂value for whole fish tissue was -0.2064. The derived time zero concentration (C_0,d) for whole fish tissues in the test item treatment group was 3.14 µg/g.

In the HCB reference group, thek₂value for whole fish tissue was -0.0387. The derived time zero concentration (C_0,d) for whole fish tissues in the HCB reference group was 1.18 µg/g.

These results are be summarized in the table below.

	Slope	Intercept	C0,d
Test item	-0.2064	1.1447	3.14
Reference item	-0.0387	0.1264	1.18
Slope=k2		C0,d(µg/g)=

 

Growth rate

Growth measurements (length and total wet weight) for individual fish collected during uptake and depuration in each condition are presented in appendix.

Growth rate constants (k_g), were calculated by performing a linear least squares correlation on the individual data of the control, treatment and reference groups.

The slopes of the linear regression were compared statistically using the student’s t-test (p = 0.05).

In each condition, there were no statistical differences between the slopes of the uptake and depuration phase growth data in the treatment group (p > 0.05) therefore the uptake and depuration data was pooled.

The growth rate constants (k_g) were -0.0093, -0.0067 and -0.0052 day^-1in the control, treatment and reference groups, respectively.

 

Food ingestion rate

The food ingestion rate constant (I) was calculated using the desired amount of food to be fed per day, weight of fish and number of fish remaining in each tank. Feeding rates were adjusted based out on the weights of sampled fish at each interval during the study. The food ingestion rate was calculated to be 0.015 g food g-1 fish day-1 (i.e. 1,5%).

 

Assimilation efficiency

The assimilation efficiency (α) is the efficiency of absorption of the test or reference substance across the gut.

 The equations and parameters used to calculate the assimilation efficiency are presented in the table below. The assimilation efficiencies for whole fish tissues in the treatment and reference group are also presented in the table below :

	a
Test item	0.0068
Reference item	0.5278
	C0,d=derived concentration at end of uptake k2=Depuration rate constant. I =Food ingestion rate constant (g food g^-1day^-1) Cfood(µg/g) = Average measured concentration in food                 t =          Days of feeding (exposure)

The assimilation efficiency for whole fish tissue in the test item treatment group was 0.0068. The assimilation efficiency for whole fish tissue in the HCB reference group was 0.5278.

 

Lipid fish content

Lipids (%)
Phase_time (days)		Control	Treatment_Y124	Reference_HCB
Uptake_0		9.5	7.9	7.9
		12.8	11.7	8.8
		8.7	6.3	11.0
	mean	10.3	8.7	9.2
	SD	2.1	2.8	1.6
Uptake_14		8.9	7.9	6.8
		9.2	13.3	8.3
		9.7	9.7	5.6
	mean	9.3	10.3	6.9
	SD	0.4	2.7	1.3
Depuration_28		7.9	3.0	11.1
		10.5	5.9	1.6
		6.9	2.9	6.3
	mean	8.4	3.9	6.3
	SD	1.8	1.7	4.7
	Mean	9.3	7.6	7.5
	SD	1.6	3.6	2.9

 

Lipid correction

The corrections were calculated using (i) the mean lipid fractions from the fish (L_fish) from Day 0 and 14 of uptake and from day 28 of depuration and (ii) The mean lipid fractions of diet (L_food) analyzed on Day 0 and 14 of uptake.

The lipid correction (L_c) factors for both the treatment and reference groups are presented in Table below.

	Test item	Reference item
Lfish	7.6 ±3.6	7.5±2.9
Lfood	14.3 ±0.4	14.3±0.2
LC	0.53	0.52

The lipid‑correction factor was calculated to be 0.53 for the treatment group and 0.52 for the reference group.

 

BMF calculations

The equations used to calculate the BMF values for the treatment and reference groups are presented in the table below:

	Test item	Reference item
k2	-0.2064	-0.0387
kg	-0.0067	-0.0052
k2g	0.1997	0.0334
BMF	0.0005	0.2048
BMFKg	0.0005	0.2367
BMFKgL	0.0010	0.4553
t1/2g	3.5	18
BMFSS	15.0	90

The growth and lipid corrected BMF (BMF_KgL) value for whole fish tissue of the test item treatment group was 0,0010. The time to reach 95% steady state (BMFSS) was 15 days and the growth corrected half-life (t1/2g) was 3,5 days.

The growth and lipid corrected BMF (BMF_KgL) value for whole fish tissue of the HCB treatment group was 0,4553. The BMF_KgLfalls within the range from the OECD ring test (0.0645 to 4.377). This suggests that dosing was properly performed, and that thePimephales promelastook up the HCB and distributed it to tissues in a manner consistent with previous studies. The time to reach 95% steady state (BMFSS) was 90 days and the growth corrected half-life (t_1/2g) was 18 days.

 

BCF estimation based on BMF values

According to the Guidance Document OECD TG 305, different methods are available to estimate the BCF from a dietary exposure.

For this, the Excel BCF Estimation Tool version 2 available on the OECD website was used (http://www.oecd.org/chemicalsafety/testing/section-3-environmental-fate-behaviour-software-tg-305.htm).

The results table the for test item, Yellow 124, is given below:

Inputs				Outputs
Variable	Value			Method 1
Mean weight at test start (g)	1,7			inputs for K1	K1	BCF Est.	Ref.
Uptake phase duration (days)	14			weight	455,12	1604,9	Hayton and Barron (1990)
Growth rate, Kg(day^-1)	-0,0067			weight	620,01	2186,4	Erickson and McKim (1990a)
Log KOW	7,12			weight	619,43	2184,4	Barber et al. (1991)
K2 g(K2- Kg)	0,1997			weight	401,86	1417,1	Barber (2003) - observed
Mean fish lipid uptake end or depuration start (fraction)	0,103			weight	637,57	2248,3	Barber (2001)
Mean fish lipid depuration end (fraction)	0,039			weight	120,86	426,2	Streit and Sire (1993)
Depuration phase duration (days)	28			weight	506,23	1785,2	Erickson and McKim (1990b)
BMFg l	0,001			weight	440,63	1553,8	Sijm et al. (1995)
				weight	544,20	1919,1	Barber (2003) - calibrated
Interim Outputs				log Kow	1149,85	4054,8	Tolls and Sijm (1995)
				log Kow	1063,26	3749,5	Spacie and Hamelink (1982)
				weight, log Kow	112,57	397,0	Hendriks et al. (2001)
Variable	Value			weight, log Kow	43,76	154,3	Thomann (1989)
Mean weight midpoint uptake phase (g)	1,678
Mean lipid content midpoint depuration phase	0,071			Method 2
K2 g l	0,284			input	Estimated K1	BCF Est.	Ref.
				K2 g l	327,35	1154,4	Brookes and Crooke (2012)
				Method 3
				input	Estimated K1	BCF Est.	Ref.
				BMFg l	12,26	43,3	Inoue et al (2012)

 

With this tool, estimated BCF ranged from 43.3 to 4054 according to the method.

In this study, This BMF_KgLestimate is reasonable (0.001), as the measured assimilation efficiency approaches zero, indicating that the test substance is not taken up from the food and the depuration rate is not really relevant.

Measured concentrations of the test substance in the feed were 374,0 µg Y124 /g , whereas maximum concentration in tissues at end of uptake was 0.182 µg Y124 /g. This would seem to indicate that test item simply does not pass through the gut lining into the tissues, and suggests that there is a large margin between the observed accumulation of the test substance and any level of accumulation that might be of concern.

Thus several lines of evidence indicate the empirical method of Inoueet.al. (2012) should be favoured, and the best available estimate of BCFKgL is 43.3.

BCF estimation was performed for the reference item using the same excel tool. The result table is given below:

Variable	Value			Method 1
Mean weight at test start (g)	1,696			inputs for K1	K1	BCF Est.	Ref.
Uptake phase duration (days)	14			weight	455,07	10321,8	Hayton and Barron (1990)
Growth rate, Kg(day^-1)	-0,005211089			weight	619,96	14061,9	Erickson and McKim (1990a)
Log KOW	5,73			weight	619,37	14048,5	Barber et al. (1991)
K2 g(K2- Kg)	0,0334			weight	401,82	9114,0	Barber (2003) - observed
Mean fish lipid uptake end or depuration start (fraction)	0,069			weight	637,52	14460,1	Barber (2001)
Mean fish lipid depuration end (fraction)	0,063			weight	120,84	2741,0	Streit and Sire (1993)
Depuration phase duration (days)	28			weight	506,17	11480,8	Erickson and McKim (1990b)
BMFg l	0,4553			weight	440,55	9992,5	Sijm et al. (1995)
				weight	543,98	12338,5	Barber (2003) - calibrated
Interim Outputs				log Kow	778,14	17649,8	Tolls and Sijm (1995)
				log Kow	664,22	15065,7	Spacie and Hamelink (1982)
				weight, log Kow	110,83	2513,8	Hendriks et al. (2001)
Variable	Value			weight, log Kow	160,00	3629,1	Thomann (1989)
Mean weight midpoint uptake phase (g)	1,679
Mean lipid content midpoint depuration phase	0,066			Method 2
K2 g l	0,044			input	Estimated K1	BCF Est.	Ref.
				K2 g l	494,84	11223,8	Brookes and Crooke (2012)
				Method 3
				input	Estimated K1	BCF Est.	Ref.
				BMFg l	302,96	6871,8	Inoue et al (2012)

 

VALIDITY CRITERIA

All the validity criteria specified in the study plan were met.

·        Water temperature variation was less than +/- 2°C in treatment, reference or control groups

·        Concentration of dissolved oxygen did not fall below 60% of the air saturation value

·        The concentration of the test item in fish food before and ate the end of the uptake phase was within a range of +/-20% (based on three replicates)

·        A high degree of homogeneity of test item in food was demonstrated. Three sample concentrations taken ate test stat did not vary more than +/- 15% from the mean.

·        Concentration of test item was not detected in control fish diet and tissue.

·        No mortality or other adverse effects was observed in both control and treatment groups.

 

Validity criteria fulfilled:

yes

Conclusions:

Fathead minnow were exposed to a control, treatment and reference diet for 14 days. Mean measured concentration of the treatment diet was 374,0 μg CI Yellow 124 /g and mean measured concentration of the reference diet was 8.2 μg HCB /g.
The growth and lipid corrected BMF value was 0.001 in analytically determined whole fish tissues in the CI Yellow 124 treatment group.
The mean measured time zero concentration (tissue concentration at Day 14 uptake) in whole fish tissue was 7,1 μg/g, while the derived time zero concentration in whole fish tissue was 3.14 μg/g suggesting the presence of undigested food in the gut tract. The time to reach 95% steady state (BMFSS) was 15 days and the estimated time to reach 50% clearance was 3,5 days.
The growth and lipid corrected BMF value for the reference substance HCB was 0,4553 in whole fish tissues. The time to reach 95% steady state (BMFSS) was 90 days and the estimated time to reach 50% clearance was 18 days.
The substance is not considered to be bioaccumulative on the basis of the study results observed.

Executive summary:

Fathead minnow were exposed to a control, treatment and reference diet for 14 days. Mean measured concentration of the treatment diet was 374.0 μg CI Yellow 124 /g and mean measured concentration of the reference diet was 8.2 μg HCB /g.

The growth and lipid corrected BMF value was 0.001 in analytically determined whole fish tissues in the CI Yellow 124 treatment group.

 

The mean measured time zero concentration (tissue concentration at Day 14 uptake) in whole fish tissue was 7.1 μg/g, while the derived time zero concentration in whole fish tissue was 3.14 μg/g suggesting the presence of undigested food in the gut tract. The time to reach 95% steady state (BMFSS) was 15 days and the estimated time to reach 50% clearance was 3, 5 days.

 

The growth and lipid corrected BMF value for the reference substance HCB was 0.4553 in whole fish tissues. The time to reach 95% steady state (BMFSS) was 90 days and the estimated time to reach 50% clearance was 18 days.

 

Results:

Conditions	Tissue type	Depuration rate constant k2g	Assimilation efficiency	Growth and lipid corrected Biomagnification factor BMFKgL
CI Yellow 124	Whole fish	0.1997	0.0068	0.0010
HCB (reference)	Whole fish	0.0334	0.5278	0.4553

  The substance is not considered to be bioaccumulative on the basis of the study results observed.

Description of key information

Results:

Conditions	Tissue type	Depuration rate constant k2g	Assimilation efficiency	Growth and lipid corrected Biomagnification factor BMFKgL
CI Yellow 124	Whole fish	0.1997	0.0068	0.0010
HCB (reference)	Whole fish	0.0334	0.5278	0.4553

 

Key value for chemical safety assessment

BMF in fish (dimensionless):

0.001

Additional information

Fathead minnow were exposed to a control, treatment and reference diet for 14 days. Mean measured concentration of the treatment diet was 374.0 μg CI Yellow 124 /g and mean measured concentration of the reference diet was 8.2 μg HCB /g.

The growth and lipid corrected BMF value was 0.001 in analytically determined whole fish tissues in the CI Yellow 124 treatment group.

 

The mean measured time zero concentration (tissue concentration at Day 14 uptake) in whole fish tissue was 7.1 μg/g, while the derived time zero concentration in whole fish tissue was 3.14 μg/g suggesting the presence of undigested food in the gut tract. The time to reach 95% steady state (BMFSS) was 15 days and the estimated time to reach 50% clearance was 3, 5 days.

The growth and lipid corrected BMF value for the reference substance HCB was 0.4553 in whole fish tissues. The time to reach 95% steady state (BMFSS) was 90 days and the estimated time to reach 50% clearance was 18 days.

 

Results:

Conditions	Tissue type	Depuration rate constant k2g	Assimilation efficiency	Growth and lipid corrected Biomagnification factor BMFKgL
CI Yellow 124	Whole fish	0.1997	0.0068	0.0010
HCB (reference)	Whole fish	0.0334	0.5278	0.4553

 The substance is not considered to be bioaccumulative on the basis of the results observed.