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Bioaccumulation: aquatic / sediment

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Endpoint:
bioaccumulation in aquatic species, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not specified, but the date of study initiation was 2007-06-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Although there is currently no guideline for this study type, the method is recognised as an animal alternative under REACH. The report provides details on the method and on the validity of the results, the procedures used are clearly described (when taking into account the articles in reference) and the results may be used in a weight of evidence approach.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: Trout S9 in vitro metabolism assay
Principles of method if other than guideline:
CE cowan Ellsberry, SD Dyer, S Erhardt, MJ Bernhard, AL Roe, ME Dowty, AV Weisbrod. 2008. Approach for extrapolating in vitro metabolism data to refine bioconcentration factor estimates. Chemosphere 70:1804-1817
GLP compliance:
yes
Remarks:
2008-05-15
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Appearance (physical state, color): Clear liquid
Molecular Formula: C17H34O4
Molecular Weight: 302.46
Radiolabelling:
no
Vehicle:
yes
Details on preparation of test solutions, spiked fish food or sediment:
The test compound (TMCH) was added to a concentration of 10 μM as a 1 mM stock prepared in ethanol using a 50 μL aliquot. Each reaction mixture was prepared as a total of 5 mL. The reaction solutions were incubated at approximately 20oC in a temperature controled shaking platform shaker over a 2 hour period
Test organisms (species):
other: trout, but species was not specified.
Details on test organisms:
The fish were supplied by the Oden State Fish Hatchery located in Alanson, MI and ranged in weight from 35 to 300 gms each. They were held for one week under standard aquaculture conditions before harvesting tissue. The fish were anesthetized with MS222 until immobile prior to sacrificing. The liver was dissected from the carcass, weighed and immediately flash-frozen in liquid nitrogen. The livers were stored at approximately -80 °C until processing further. The trout livers were, minced, and mixed with buffer in a 4:1 ratio of buffer to liver (v:w). The buffer (pH 7.4) was composed of 50 mM potassium phosphate, 0.15 M potassium chloride, and 0.2 M sucrose. The tissue was homogenized on ice using Teflon tipped pestle with glass
mortar. The homogenate was placed into chilled centrifuge tubes and centrifuged at 12,000 g for 30 min at 1-3°C. The supernatant (S9 fraction) was carefully removed and transferred to a chilled bottle or beaker and frozen in 2 mL aliquots on dry ice and transferred to an -80 °C freezer for storage. The total protein was determined using the Pierce BCA™ method (Pierce, Rockford, IL).
Route of exposure:
aqueous
Test type:
not specified
Water / sediment media type:
not specified
Total exposure / uptake duration:
120 min
Details on test conditions:
Heat deactivated control reaction solutions were prepared in triplicate with trout liver S9 fraction inactivated by placing a 50 mL centrifuge tube of the diluted S9 preparation in boiling water for approximately 5 minutes. Data generated by the incubation of the heat deactivated S9 with the test substance were used to evalaute for any non-biological dissipation processes such as binding or hydrolysis. A single deactivated reaction solution containing no TMCH was also prepared to evaluate matrix interference during the analysis. Aliquots of 0.5 mL were removed from each reaction solution containing active S9 at 0, 20, 45, 60, 90 and 120 minutes. Aliquots of 0.5 mL were removed from the reaction solutions containing heat-deactived S9 and no test materials at 0, 60, and 120 minutes. The aliquots were added to an equal volume (0.5 mL) of ice-cold hexane and placed on an ice-bath for 30 minutes. Precipitated proteins were removed from the solution by centrifugation.
Nominal and measured concentrations:
Nominal concentration : 10 µM
Reference substance (positive control):
no
Details on estimation of bioconcentration:
The BCF was predicted based on the mass-balance model described by Arnot and Gobas (2003, 2004). To extrapolate from intrinsic clearance rates in the in vitro tests to the kMET, which is used to refine the predicted BCF value, a number of physical and physiological parameters describing fish are required.
Type:
BCF
Value:
>= 442.73 - <= 765.76 L/kg
Basis:
other: in vitro extrapolation
Calculation basis:
kinetic
Details on results:
body rate constant of metabolism (kMET) using the technique described by Cowan et al.,(2007). The kMET was then used as input in the Gobas bioaccumulation model to provide an estimate of metabolism when calculating BCF. The original BCF for TMCH calculated using the Gobas model and assuming no metabolism was 46,097 kg/day. With the addition of metabolism BCF ranged from 422 to 765 kg/day. The BCF with the addition of metabolism is significantly lower than 2000 and would be expected to not be considered bioaccumulative using regulatory criteria.
Reported statistics:
Descriptive statistics were used, i.e., mean ± standard deviation. All calculations in the data were conducted using Microsoft Excel spreadsheets and data in full precision mode (15 digits of accuracy).

kMET Calculation

Trout w/ arterial

hepatic blood flow

Trout with arterial and

portal hepatic blood

flow

CLm (ml/hr/gm of protein)

8.67E+01

8.67E+01

CLi -- Intrinsic Clearance in liver (ml/hr/Kg)

5.96E+01

5.96E+01

CLi -- Intrinsic Clearance in liver (L/d/Kg)

1.43E+00

1.43E+00

CLh -- Hepatic Clearance

incorporating blood flow (L/d/Kg))

7.18E-01

1.25E+00

kMET(1/day)

0.128

0.224

 

BCF Calculation

 

 

K1 - uptake rate (L/Kg day)

1.00E+02

1.00E+02

K2- rate of elimination via resp

(1/day)

2.75E-05

2.75E-05

Ke - rate of chemical egestion via fecal material (kg/kg/day)

1.35E-03

1.35E-03

Kg - growth dilution (1/day)

7.96E-04

7.96E-04

BCF = k1/(k2+ke+kg+km) incorporating kMET

765.76

442.73

BCF with kMET= 0

46097.54

46097.54

Validity criteria fulfilled:
not applicable
Conclusions:
The BCF value of the substance was determined with and without metabolism being taken into account. The calculation is based on the Arnot Gobas BCF mass balance model run with and without metabolism. When metabolic rate (Kmet) is set to 0, the model calculates a BCF ot 46097. When Kmet is experimentally determined using two methods: arterial hepatic and arterial hepatic and portal, blood flow extrapolation further to a trout hepatocyte in vitro study, BCFs are calculated as 766 and 443 L/Kg respectively.
Executive summary:

The rate of fish metabolism for the organic peroxide 1,1-di(t-butylperoxy)3,3,5-

trimethylcyclohexane (TMCH) was determined in vitro using a trout liver S9 fraction.

Each reaction mixture contained 2 mg/mL S9 protein and the concentration of TMCH

was 10μM. The experiment was conducted in triplicate, with controls containing heat

deactivated S9 protein and 10μM TMCH. Sub-samples were removed over the course of

2 hours to determine the rate of degradation due to metabolism and abiotic processes.

The reaction was halted by the addition of an equal volume of cold hexane to each sub

sample, the solutions thoroughly vortexed and centrifuged to separate phases. The upper

hexane layer was removed and analyzed by GC/MS to determine the concentration of the

peroxide. First order kinetics was assumed and the % parent remaining was plotted

against time and fitted to an exponential curve. Kinetics were determined for both

biologically active and deactivated control reaction mixtures. The biologically active rate

constant was determined by subtracting the rate constant determined for the deactivated

control solution from the rate constant determined for the biologically active solution.

This rate constant was used to calculate the in vitro half-life of metabolism for TMCH of

173 minutes. The half life was used to calculate a rate of metabolism of 0.87 (μmoles

parent/gm protein/ hour). The in vitro rate of metabolism was extrapolated to a whole body rate constant of metabolism (kMET) using the technique described by Cowan et al.,. (2007). The in vivo rate constants of metabolism or kMET were calculated as 0.129 or 0.224 /day.When metabolic rate (Kmet) is set to 0, the model calculates a BCF ot 46097.

When Kmet is experimentally determined and the values obtained from the two methods incorporated into the equation (arterial hepatic and arterial hepatic and portal blood flow) further to a trout hepatocytein vitrostudy, BCFs are calculated as 766 and 443 L/Kg respectively.

Endpoint:
bioaccumulation in aquatic species, other
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not specified, but the date of study initiation was 2008-02-29
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Good quality study with GLP statement and substance identity showing the impact of contact time with fish tissue on the recovery of the organic peroxide CAS#6731 -36 -8 and the production of degradation products.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Remarks:
2008-11-12
Radiolabelling:
no
Vehicle:
yes
Test organisms (species):
other: rainbow trout, but specie was not specified
Route of exposure:
aqueous
Test type:
not specified
Water / sediment media type:
not specified
Total exposure / uptake duration:
60 min
Nominal and measured concentrations:
Nominal concentrations : 10 and 1 µg/g of CAS#6731-36-8
Measured concentrations : The concentration of CAS#6731-36-8 in samples spiked at 10 µg/g ranged from a maximum of 10.56 µg/g at T=0min to a minimum of 6.41 µg/g at 45 minutes. The concentration of CAS#6731-36-8 gin sample spiked at 1.0 µg/g varied more widely than those dosed at 10 µg/g; the concentration ranged from a maximum of 1.14 µg/g at T=0min to a minimumof 0.49 µg/g at 60 minutes. See table 1 in "Any other information on results incl. tables" field.
Details on estimation of bioconcentration:
No estimation of bioconcentration was performed during the study. However, the objective of this study was to evaluate the impact of contact time on the recovery of the organic peroxide CAS#6731-36-8 from freshly prepared and potentially metabolically active fish tissue.

Table 1 : Concentrations of CAS#6731 -36 -8, CAS#873 -94 -9 and t-butanol in fish tissue samples *

Sample number

Spike level, time (min.) and replicate

CAS#6731-36-8 (µg/g)

CAS#6731-36-8 % recovery

CAS#873-94-9 (µg/g)

t-butanol (µg/g)

1

2

5

6

Control-0-1

Control-0-2

Control-60-1

Control-60-2

<LLQ**

<LLQ

<LLQ

<LLQ

--***

--

--

--

--

--

--

--

<LLQ

<LLQ

<LLQ

<LLQ

Samples spiked with CAS#6731-36-8

7

8

9

10

11

12

13

14

15

16

 

17

18

19

20

21

22

23

24

25

26

10 µg/g 0-1

10 µg/g 0-2

10 µg/g 10-1

10 µg/g 10-2

10 µg/g 30-1

10 µg/g 30-2

10 µg/g 45-1

10 µg/g 45-2

10 µg/g 60-1

10 µg/g 60-2

 

1 µg/g 0-1

1 µg/g 0-2

1 µg/g 10-1

1 µg/g 10-2

1 µg/g 30-1

1 µg/g 30-2

1 µg/g 45-1

1 µg/g 45-2

1 µg/g 60-1

1 µg/g 60-2

10.56

10.08

8.64

9.44

9.60

8.96

6.41

10.00

9.92

10.24

 

0.88

1.14

1.06

0.87

0.57

0.54

0.67

0.90

0.49

0.96

105.6

100.8

86.4

93.6

96.0

88.8

63.9

99.2

98.4

102.4

 

88.0

114.4

105.6

86.4

57.0

54.2

66.6

88.8

49.0

96.0

0.23

0.17

<LLQ

0.23

0.20

0.16

0.13

0.30

0.29

0.29

 

0.02

0.03

0.03

0.02

<LLQ

<LLQ

<LLQ

<LLQ

<LLQ

<LLQ

2.77

<LLQ

<LLQ

1.07

<LLQ

<LLQ

1.85

<LLQ

<LLQ

0.97

 

<LLQ

<LLQ

<LLQ

<LLQ

<LLQ

<LLQ

<LLQ

<LLQ

<LLQ

<LLQ

Samples spiked with t-butanol

27

28

29

30

31

32

5 µg/g 0-1

5 µg/g 0-2

5 µg/g 30-1

5 µg/g 30-2

5 µg/g 60-1

5 µg/g 60-2

--

--

--

--

--

--

N/A****

N/A

N/A

N/A

N/A

N/A

--

--

--

--

--

--

1.29

<LLQ

<LLQ

<LLQ

<LLQ

<LLQ

* tert-butanol (purity of 99.5%) and CAS#873 -94 -9 / 3,3,5 -trimethylcyclohexane (purity of 96.4%) were used for identification and quantification of degradation products anticipated from the degradation of CAS#6731 -36 -8.

** <LLQ = <10.4 ng/mL for CAS#6731 -36 -8, <1.08 ng/mL for CAS#873 -94 -9 and <10.7 ng/mL for t-butanol

*** -- = not analyzed for specific compound

**** N/A = not applicable

Validity criteria fulfilled:
not applicable
Conclusions:
When spiking freshly prepared fish homogenate with the organic peroxide CAS#6731-36-8 at residue levels, care should be taken to extract the tissues as quickly as possible. The lower extraction efficiencies could be attributed to the metabolism of CAS#6731-36-8 by peroxidases still active in the tissues. The detection of t-butanol may also be limited as a result of metabolism as well as tissues, especially those associated with the liver have elevated levels of alcohol-dehydrogenase.
Executive summary:

The objective of this study was to evaluate the impact of contact time on the recovery of the organic peroxide CAS#6731 -36 -8 from freshly prepared and potentially metabolically active fish tissue. Samples of freshly homogenized rainbow trout were spiked with the organic peroxide CAS#6731 -36 -8 at either 10 µg/g or 1.0 µg/g. In addition, samples were also spiked at 5 µg/g with t-butanol, a known metabolite of CAS#6731 -36 -8. For comparison tissue samples were prepared with only carrier solvent (100% ethanol) to evaluate for matrix interferences. The samples were allowed to sit on the bench top with samples removed in duplicate over the course of 1 hour. At each sampling interval, 20 mL of acetonitrile was added to the tissue. The tissue were thoroughly homogenized with the solvent and centrifuged to pellet the tissue. Aliquots were removed from the extraction solvent concentrated to dryness and re-suspended in either ethyl acetate for analysis of the peroxide or hexane for analysis of the t-butanol. The concentration of CAS#6731 -36 -8 in samples spiked at 10 µg/g nominal ranged from a maximum of 10.56 µg/g at T0 to a miminum of 6.41 µg/g at 45 minutes. When normalized to T0 recovery varied from 100% to a minimum of 79.5%. Samples spiked at 1.0 µg/g (nominal) showed more variability and ranged from 1.14 µg/g to a minimum of 0.49 µg/g at 0 and 60 minutes, respectively. When normalized to the average concentration at T0, sample recovery ranged from an average of 100% at T0 to a minimum of 49% at 60 minutes. In general, samples spiked at 1.0 µg/g showed more extreme losses of recovery with incubation time, which were likely due to the presence of metabolically active tissues in the freshly prepared fish homogenate. This study demonstrates the importance when working with metabolically labile materials at trace levels, to work quickly with the tissues to minimize the potential impact of metabolism on the organic peroxide. Samples were also analyzed for t-butanol, a proposed primary degradation product. When spiked at 5 µg/g in fish tissue, all samples had less than the limit of quantification other than a single sample at T0. Four out of twelve samples spiked with 10 µg/g CAS#6731 -36 -8 had detectable quantities of t-butanol while no samples spiked at 1 µg/g CAS#6731 -36 -8 had detectable quantites. Liver and other tissues are known to have significant levels of alcohol dehydrogenase, which would quickly metabolize the alcohol to its concomitant aldehyde.

Description of key information

For this substance, an in vitro study is used in a weight of evidence approach in an attempt to quantitatively assess the BCF. The in vivo studies cannot be used for risk assessment due to major methodological deficiencies (notably the BCF was tested using aqueous concentrations 10 to 100 time higher than the solubility limit). The most appropriate existing study is the Trout S9 in vitro metabolic study used in conjunction with a BCF model which is considered as weak evidence of non B. This provides BCF values of 433 to 766 L/Kg. However, no completely valid study on BCF exists to date.

Key value for chemical safety assessment

BCF (aquatic species):
766 L/kg ww

Additional information

The bioaccumulation test in CarpCyprinus carpio was conducted on the reference substance in a NITE study. The test substance concentrations were 0.2 and 0.02 mg/L maintained in a solvent. The measured concentrations within the test water was an established value of 60% or greater. The duration of exposure was 8 wk, with continuous water flow-through method. A Bioconcentration factor (BCF) at 6510 -6200 for the 1st concentration section (0.2 mg/L) and 4960-5970 for the 2nd concentration section (0.02 mg/L), based on steady state values. No kinetics were provided, nor tissue concentrations. This in vivo study is thought to be fundamentally flawed as the test substance appears to diminish relatively rapidly in water and more so in fish tissues. This can be attributed to the fact that the test was performed at water concentrations 10 and 100 times the estimated water solubility of the test substance.

Moreover, critical information on the relevance of time related recovery factors and analytical methodology data are missing. The study cannot be considered valid.

An in vitro study was performed using the trout S9 in vitro metabolism assay (Erhardt 2008) and the metabolism results were combined with a BCF uptake depuration model (Arnott & Gobas, 2003, 2004). When metabolic rate (Kmet) is set to 0, the model calculates a BCF of 46097. When Kmet is experimentally determined and the values obtained from the two methods incorporated into the equation (arterial hepatic and arterial hepatic and portal blood flow) further to a trout hepatocyte in vitro study, BCFs are calculated as 766 and 443 L/Kg respectively.

In the absence of a valid in vivo study the BCF value of 766 L/Kg will be used for risk assessment as a worst case estimate.