4-(4-nitrophenylazo)-2,6-di-sec-butyl-phenol

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:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Bioconcentration Factors derived from Biomagnification Factors are highly conservative because to determine the BCF it is assumed that the test substance is completely soluble in the water phase. For Mortrace™ SB TGAI this assumption is not valid because Mortrace™ SB TGAI is not water soluble to any appreciable degree. Therefore, the calculated BCF values are highly conservative estimates and possibly inappropriate for use in standard environmental fate models.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)

Deviations:

no

Remarks:

The reference substance, Hexachlorobenzene was not characterized in accordance with Good Laboratory Practice standards

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1730 (Fish Bioconcentration Test)

Deviations:

no

Remarks:

same as above

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms: Fish were sampled on days 0 and 14 of uptake and days 0, 1, 2, 4, 7, 14 and 21 of depuration. Fish samples were collected from each test chamber using a soft net. Ten fish were impartially sampled in a nonsystematic manner from each control chamber on day 0 of uptake and each control and treated chamber on day 14 of uptake, where five fish were collected for whole fish analyses and the remaining five fish were dissected to yield fillet tissue (i.e., tissue without internal organs) and viscera tissue (i.e., internal organs) for analysis of Mortrace™ SB TGAI and HCB residues. Ten fish were impartially sampled in a nonsystematic manner from each control chamber and treated chamber on days 1 and 7 of depuration, where five fish were collected for whole fish analyses and the remaining five fish were dissected to yield fillet tissue and viscera tissue for analysis of Mortrace™ SB TGAI residues. Five fish were impartially sampled in a nonsystematic manner from each control chamber and treated chamber on days 2, 4, 14 and 21 of depuration for analysis of Mortrace™ SB TGAI residues in whole fish. Two fish were impartially sampled in a nonsystematic manner from each control chamber on day 0 of uptake and from each control and treated chamber on day 0 of depuration for determination of lipid fractions in whole fish.
The collected fish were sacrificed instantly by cervical dislocation, weighed, measured. All individuals were measured for total length using a millimeter scale and blotted wet weight using an electronic balance. Following sampling, each fish tissue sample was immediately placed into individual labeled polyethylene containers with liquid nitrogen. The containers were labeled with the following: 1) ABC study number, 2) sampling date, 3) sampling day, 4) study phase (i.e., uptake or depuration), 5) matrix (i.e., whole fish, fillet, or viscera), 6) treatment group (i.e., control or treated), 7) fish sample number, and 8) test chamber replicate. Fish tissue samples were stored at approximately -20°C, following evaporation of liquid nitrogen, until processed for analysis. Frozen samples were processed to a fine powder using a Polytron and liquid nitrogen.
Survival was monitored daily by visually inspecting each test chamber, and any behavioral or physical changes were recorded, including abnormalities. The test chambers were cleaned periodically to minimize biological growth on the sides and bottom of the test chamber. After 21 days of depuration, the remaining non-sampled fish were removed from each replicate chamber, euthanized with tricaine methanesulfonate (MS-222; Western Chemical, Inc.), and then measured and weighed.

- Sampling intervals/frequency for test medium samples: A primary stock solution of Mortrace™ SB TGAI (Lot No.: RWS2B-62) was prepared on May 24, 2005, at 1.07 mg total product/mL in toluene and was stored at room temperature. Dilutions of the primary stock solution were prepared in toluene on May 24, June 7, July 28, August 19, August 26, and November 8, 2005, and were stored at room temperature. The primary stock solution and dilutions were used as the analytical standards or matrix spiking solutions for quality control (QC) samples during the validation of the analytical method for whole fish tissue, determination of range-finding test feed homogeneity, analysis of rangefinding test tissue samples, and determination of range-finding test feed stability.
A primary stock solution of Mortrace™ SB TGAI (Lot No.: ZA78743-5-2S) was prepared on February 3, 2006, at a concentration of 0.941 mg a.i./mL in toluene and was stored in an amber bottle at room temperature. Dilutions of the primary stock solution were prepared in acetonitrile on February 4, 2006, and were stored in the dark at room temperature. The dilutions were used to prepare analytical standards for the analysis of tissue and feed during the definitive test.
A second primary stock solution of Mortrace™ SB TGAI (Lot No.: ZA78743-5-2S) was prepared on February 3, 2006, at a concentration of 0.772 mg a.i./mL in toluene and was stored in an amber bottle at room temperature. Dilutions of the primary stock solution were prepared in toluene on February 3, 2006, and were stored an amber bottle or in the dark at room temperature. The dilutions were used to prepare matrix (i.e., feed and tissue) spiking solutions for QC samples during the analysis of tissue and feed during the definitive test.
A primary stock solution of HCB was prepared on May 18, 2005, at a concentration of 0.559 mg a.i./mL in toluene and was stored at room temperature. Dilutions of the primary stock solution were prepared in toluene on May 23, August 27, August 29, and November 7, 2005, and were stored at room temperature. The primary stock solution and dilutions were used as the analytical standards or matrix spiking solutions for QC samples during the validation of the analytical method for whole fish tissue and feed, determination of rangefinding test feed homogeneity, analysis of range-finding test tissue samples, and
determination of range-finding test feed stability as well as the analytical standards or matrix spiking solutions for QC samples during the analysis of tissue and feed during the definitive test.
A second primary stock solution of HCB was prepared on October 7, 2005, at a concentration of 2.48 mg a.i./mL in acetone and was stored at room temperature. Dilutions of the primary stock solution were prepared in toluene on February 7, 2006, and were stored at room temperature. The dilutions of the primary stock solution were used as the analytical standards during the analysis of tissue and feed during the definitive test.

- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods): Fish tissue and feed samples analyzed during the definitive test were prepared in the following manner:
Approximately 1 g samples were weighed into 15-mL capacity culture tubes. Samples were extracted with 10 mL toluene on an Eberbach reciprocating shaker on high setting for 10 minutes. After shaking, the samples were centrifuged on an IEC clinical centrifuge (tabletop model) at the highest setting (e.g., 7) for 10 minutes. Nine-milliliters were withdrawn from each sample and percolated through a Varian Mega Bond Elut SPE cartridge (bonded phase SI, 6 cc/1 g). The cartridges were washed prior to the sample application with 8 mL toluene. After sample application, the cartridges were further rinsed with 6 mL toluene. Samples were then brought to 15 mL volume with toluene. Dilutions were carried out with toluene for HCB analyses or acetonitrile for MortraceTM SB TGAI analyses. QC samples were prepared in a similar manner on the same day of analysis.
HCB residues were measured in all tissue (i.e., whole fish, fillet and viscera) collected only on days 0 and 14 of uptake to evaluate the assimilation efficiency of the treated diet and was not measured thereafter through depuration.
Lipids were extracted from whole fish and feed samples for calculation of percentage of lipid content using the Bligh and Dyer method (5) in the following manner:
1) A weighed sample was blended in a mixture of chloroform : methanol ( in the ratio 1:2), with the water in this step at the proportion 0.8 parts. Note: The
organic solvent portions were adjusted, or water added to give a beginning ratio of chloroform: methanol: water in sample of 1:2:0.8.
2) Additional chloroform was added to adjust the chloroform: methanol ratio to 2:2, followed by further blending.
3) Distilled water was added to mixture to achieve ratio of 2 chloroform:2 methanol: 1.8 water, followed by further blending.
4) Final blended portions were filtered through filter paper and filtrate collected.
5) The tissue residue (on filter paper) and filter paper was blended with 1 part chloroform and then filtered through filter paper using the original filter funnel and the collected filtrate was combined with initial filtrate.
6) The blender, funnel with paper, and post extract sample were rinsed with 0.5 part chloroform and combined with the initial filtrate.
7) The filtrate was allowed to separate and clarify.
8) The methanol layer (top layer) of the filtrate was removed, even at the cost of a small portion of the chloroform layer.
9) The volume of the filtrate chloroform layer was measured.
10) A measured portion or the entire chloroform layer was transferred to a tarred container and the chloroform was evaporated under gentle N2 stream.
11) The weights of dried residue and container were recorded.
12) After weighing, the residue was re-suspended in additional chloroform to detect presence of non-lipid material (i.e. insoluble material). No non-lipid material was present.
13) Lipid content of the sample was accordingly calculated.

Vehicle:

yes

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

Range finding test:
The treated diet was prepared October 7, 2005, by dosing 350 g of Salmon Starter #2 (ABC Lot No. SS0405) with a dosing solution containing a mixture of 0.7 mg Mortrace™ SB TGAI and 35 mg hexachlorobenzene (HCB) in 35 mL acetone. The entire 35 mL dosing solution volume was added to 350 g of feed, 5 mL at a time, and the feed was vigorously mixed within a half-gallon glass jar following each 5 mL addition. A final 5 mL addition consisted of an acetone rinse of the dosing solution mixing chamber; therefore the total volume of acetone applied to feed was 40 mL. The resulting nominal concentration of the treated diet was 2 μg Mortrace™ SB TGAI/g and 100 μg HCB/g. Following the final 5 mL addition and final mixing, the contents of the jar were evenly spread onto a foil-lined metal pan. The pan with its contents was placed in a fume hood and the acetone was allowed to evaporate completely. After all acetone evaporated and the dosed feed was dry (i.e., approximately 4 hours), the entire contents of the pan were transferred to a quart-glass jar. The prepared treated diet was stored in the dark at room temperature.
The acetone-dosed control diet was prepared October 13, 2005, by dosing 350 g of Salmon Starter #2 (ABC Lot No. SS0405) with 40 mL acetone, 5 mL at a time, and the feed was vigorously mixed within a half-gallon glass jar following each 5 mL addition. Following the final 5 mL addition and final mixing, the contents of the jar were evenly spread onto a foil-lined metal pan. The pan with its contents was placed in a fume hood and the acetone was allowed to evaporate completely. After all acetone evaporated and the control feed was dry (i.e., approximately 3 hours), the entire contents of the pan were transferred to a quart-glass
jar. The prepared control diet was stored in the dark at room temperature.

Definitive test:
The treated diet was prepared February 2, 2006, by dosing 350 g of Salmon Starter #2 (ABC Lot No.: SS010406) with a dosing solution containing a mixture of 0.7 mg Mortrace™ SB TGAI and 35 mg hexachlorobenzene (HCB) in 35 mL acetone. The entire 35 mL dosing solution volume was added to 350 g of feed, 5 mL at a time, and the feed was vigorously mixed within a half-gallon glass jar following each 5 mL addition. A final 5 mL addition consisted of an acetone rinse of the dosing solution mixing chamber; therefore the total volume of acetone applied to feed was 40 mL. The resulting nominal concentration of the
treated diet was 2 μg Mortrace™ SB TGAI/g and 100 μg HCB/g. Following the final 5 mL addition and final mixing, contents of the jar were evenly spread onto a foil-lined metal pan. The pan with its contents was placed in a fume hood and the acetone was allowed to evaporate completely. After all acetone evaporated and the dosed feed was dry (i.e., approximately 3 hours), the entire contents of the pan were transferred to a quart-glass jar. The prepared treated diet was stored in the dark at room temperature.
The acetone-dosed control diet was prepared by dosing 350 g of Salmon Starter #2 (ABC Lot No. SS010406) with 40 mL acetone, 5 mL at a time, and the feed was vigorously mixed within a half-gallon glass jar following each 5 mL addition. Following the final 5 mL addition and final mixing, contents of the jar were evenly spread onto a foil-lined metal pan. The pan with its contents was placed in a fume hood and the acetone was allowed to evaporate completely. After all acetone evaporated and the control feed was dry (i.e., approximately 2 hours), the entire contents of the pan were transferred to a quart-glass jar. The prepared control diet was stored in the dark at room temperature.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Rainbow trout (Oncorhynchus mykiss) were obtained from Trout Lodge, Sumner, Washington. The trout were received as ‘swim-up’ age on December 8, 2005. Fish were fed salmon starter daily while maintained at approximately the test temperature in ABC Laboratories’ culture facility. No diseases were observed or treated during the 14 day period prior to use in the test. Mortality was <10% during the 7 day period immediately prior to initiation of the definitive test.

Route of exposure:

feed

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

14 d

Total depuration duration:

21 d

Hardness:

140 to 154 mg CaCO3/L

Test temperature:

13.5 to 14.7°C

pH:

7.7 to 8.4

Dissolved oxygen:

8.2 to 11.9 mg/L (83 to 120% saturation)

TOC:

no data

Salinity:

no data

Details on test conditions:

Range finding test:
The in-life phase of the range-finding test was performed from October 14 to 31, 2005. The fish were fed daily during the test and were observed for mortality and abnormalities each day.
Forty juvenile rainbow trout (mean wet weight of 3.3 g/fish based on a sub-sample of five fish) were placed into both control and treated test chambers. Control fish were offered the acetone-dosed control diet and the treated fish were offered the treated diet at a level of approximately 1.5% of body weight twice each day (i.e., total of 3% of body weight each day) starting on day 0 and ending on day 9 of the uptake phase. Uneaten food and feces were siphoned from the test chambers approximately 30 to 60 minutes after each feeding. Diets were not offered prior to sampling (i.e., approximately 21 hours post-feeding) of control and treated fish on day 10 of uptake.
The depuration phase was initiated immediately following the sampling of fish on day 10 of uptake. The depuration phase continued for a period of seven days. During depuration, fish in the control and treated test chambers were offered a diet of non-treated, non-acetone dosed Salmon Starter #2 (ABC Lot No.: SS0405). Control and treated fish were offered the diet at a level of approximately 3.0% of body weight on day 0 of depuration and 1.5% of body weight twice each day (i.e., total of 3% of body weight each day) starting on day 1 of depuration and ending on day 6 of depuration. Uneaten food and feces were siphoned from the test chambers approximately 30 to 60 minutes after each feeding. Visual estimations of portion of offered diet consumed, performed approximately 30 to 60 minutes following offering of diet, indicated 90% of the diet was consumed at each offering during the depuration phase. The diet was not offered prior to sampling (i.e., approximately 19 hours post-feeding) of control and treated fish on day 7 of depuration.
Fish were sampled on day 10 of uptake and day 7 of depuration. Fish samples were collected from each test chamber using a soft net. Twenty fish were impartially sampled in a nonsystematic manner from the control chamber. The collected fish were sacrificed instantly by cervical dislocation, weighed, measured. Ten of the twenty fish sampled were collected for whole fish analyses and the remaining ten were dissected to yield fillet tissue (i.e., tissue without internal organs) and viscera tissue (i.e., internal organs). Following sampling, each fish tissue sample was immediately placed into individual appropriately labeled polyethylene containers with liquid nitrogen. The same process was also used for 20 fish removed from the treated chamber. Fish tissue samples were stored at approximately -20°C until processed for analysis. Frozen samples were processed to a fine powder using a
Polytron and liquid nitrogen.

Definitive study:
The in-life phase of the definitive test was performed from February 9 to March 16, 2006. The fish were fed daily during the test and were observed for mortality and abnormalities each day.
A total of 284 juvenile rainbow trout were impartially selected from the culture population on February 6, 2006. The fish were then impartially transferred five at a time to each of four test chambers (i.e., duplicate control and treated test chambers) using a soft net. The addition of five fish was repeated until each aquarium contained the appropriate number of fish. Each control chamber each contained 77 fish and each treated chamber contained 65 fish. The fish were maintained in the test chambers under test conditions. Following placement of fish, ten additional fish were impartially selected from the culture population and used to establish the initial fish mean weight. The rainbow trout in each test chamber were offered a diet of nontreated, non-acetone dosed Salmon Starter #2 (ABC Lot No.: SS010406) at a level of approximately 3.0% of body weight on February 6, 2006, and 1.5% of body weight twice each day (i.e., total of 3% of body weight each day) on February 7 and 8, 2006. Visual estimations of portion of offered diet consumed, performed approximately 30 to 60 minutes following offering of diet, indicated 20% of the diet was consumed following the first two offerings and 100% of the diet was consumed following the remaining offerings. Uneaten food and feces were siphoned from the test chambers approximately 30 to 60 minutes after each feeding. The diet was not offered prior to sampling (i.e., approximately 18 hours postfeeding) of control on day 0 (February 9, 2006) of uptake. This three day holding period was performed to allow the fish to acclimate to the exposure system.
Beginning on day 0 and ending on day 13 of the uptake phase, control fish were offered the acetone-dosed control diet and the treated fish were offered the treated diet (i.e., 2 μg Mortrace™ SB TGAI/g and 100 μg HCB/g) at a level of approximately 1.5% of body weight twice each day (i.e., total of 3% of body weight each day). Uneaten food and feces were siphoned from the test chambers approximately 30 to 60 minutes after each feeding. Diets were not offered prior to sampling (i.e., approximately 18 hours post-feeding) of control and treated fish on day 14 of uptake. On day 7 of the uptake phase, a sub-sample of five fish from each replicate chamber was weighed to determine body weight for control and treated fish. The amount fed was re-calculated, based on the weights of the fish collected in an effort to maintain consistent body weight and lipid content. The weighed fish were returned to their original test chamber after weighing.
The depuration phase was initiated immediately following the sampling of fish on day 14 of uptake. Beginning with the replicate A control uptake test chamber, all fish were transferred with a soft net to the corresponding control depuration chamber. Transfer of fish from replicate B control uptake test chamber was then performed followed by transfer of fish from replicates A and B treated uptake test chambers. The depuration phase continued for a period of 21 days.
During depuration, fish in the control and treated test chambers were offered a diet of nontreated, non-acetone dosed Salmon Starter #2 (ABC Lot No.: SS010406). Control and treated fish were offered the diet at a level of approximately 1.5% of body weight twice each day (i.e., total of 3% of body weight each day) starting on day 0 of depuration and ending on day 20 of depuration. Uneaten food and feces were siphoned from the test chambers approximately 30 to 60 minutes after each feeding. Visual estimations of portion of offered diet consumed, performed approximately 30 to 60 minutes following offering of diet,
indicated 40 to 100% of the diet was consumed at each offering during the depuration phase. The diet was not offered approximately 16 to 20 hours prior to sampling of control and treated fish on days 1, 2, 4, 7, 14 and 21 of depuration. The amount fed was re-calculated, based on the weights of the fish collected at preceding sampling intervals, in an effort to maintain consistent body weight and lipid content.
Fish were sampled on days 0 and 14 of uptake and days 0, 1, 2, 4, 7, 14 and 21 of depuration. Fish samples were collected from each test chamber using a soft net. Ten fish were impartially sampled in a nonsystematic manner from each control chamber on day 0 of uptake and each control and treated chamber on day 14 of uptake, where five fish were collected for whole fish analyses and the remaining five fish were dissected to yield fillet tissue (i.e., tissue without internal organs) and viscera tissue (i.e., internal organs) for analysis of Mortrace™ SB TGAI and HCB residues. Ten fish were impartially sampled in a nonsystematic manner from each control chamber and treated chamber on days 1 and 7 of depuration, where five fish were collected for whole fish analyses and the remaining five fish were dissected to yield fillet tissue and viscera tissue for analysis of Mortrace™ SB TGAI residues. Five fish were impartially sampled in a nonsystematic manner from each control chamber and treated chamber on days 2, 4, 14 and 21 of depuration for analysis of Mortrace™ SB TGAI residues in whole fish. Two fish were impartially sampled in a nonsystematic manner from each control chamber on day 0 of uptake and from each control and treated chamber on day 0 of depuration for determination of lipid fractions in whole fish.
The collected fish were sacrificed instantly by cervical dislocation, weighed, measured. All individuals were measured for total length using a millimeter scale and blotted wet weight using an electronic balance. Following sampling, each fish tissue sample was immediately placed into individual labeled polyethylene containers with liquid nitrogen. The containers were labeled with the following: 1) ABC study number, 2) sampling date, 3) sampling day, 4) study phase (i.e., uptake or depuration), 5) matrix (i.e., whole fish, fillet, or viscera), 6) treatment group (i.e., control or treated), 7) fish sample number, and 8) test chamber replicate. Fish tissue samples were stored at approximately -20°C, following evaporation of liquid nitrogen, until processed for analysis. Frozen samples were processed to a fine powder using a Polytron and liquid nitrogen.
Survival was monitored daily by visually inspecting each test chamber, and any behavioral or physical changes were recorded, including abnormalities. The test chambers were cleaned periodically to minimize biological growth on the sides and bottom of the test chamber. After 21 days of depuration, the remaining non-sampled fish were removed from each replicate chamber, euthanized with tricaine methanesulfonate (MS-222; Western Chemical, Inc.), and then measured and weighed.

Nominal and measured concentrations:

2 μg Mortrace™ SB TGAI and 100 μg Hexachlorobenzene/g

Reference substance (positive control):

yes

Remarks:

Hexachlorobenzene

Details on estimation of bioconcentration:

Calculations of Mortrace™ SB TGAI residues were performed by the standard analysis function of Chemstation software combined with an Excel spreadsheet. Calculations of HCB residues were performed by external standard analysis using values derived from MULTICHROM software (Version 2.3).

Lipid content:

3.8 - 6 %

Time point:

start of exposure

Remarks on result:

other: The fraction of lipids in the control whole fish at day 0 of uptake ranged from 3.8 to 6.0% (mean ±SD: 5.0 ±1.1).

Lipid content:

4.9 - 5.8 %

Time point:

start of exposure

Remarks on result:

other: The fraction of lipids in the control whole fish at day 0 of depuration ranged from 4.9 to 5.8% (mean ±SD: 5.0 ±0.47).

Lipid content:

5.9 - 7 %

Time point:

other: day 0 of depuration

Remarks on result:

other: The fraction of lipids in the treated whole fish at day 0 of depuration ranged from 5.9 to 7.0% (mean ±SD: 6.0 ±0.55).

Type:

BCF

Value:

842 - 1 070 dimensionless

Basis:

other:

Calculation basis:

other:

Remarks on result:

other: Conc.in environment / dose:2.22 (±0.0656) μg Mortrace™ SB TGAI/g in feed

Type:

BMF

Value:

0.006 dimensionless

Basis:

other:

Calculation basis:

other:

Remarks on result:

other: Conc.in environment / dose:2.22 (±0.0656) μg Mortrace™ SB TGAI/g in feed

Type:

other: Lipid-normalized biomagnification factor BMFL

Value:

0.017 dimensionless

Basis:

other:

Calculation basis:

other:

Remarks on result:

other: Conc.in environment / dose:2.22 (±0.0656) μg Mortrace™ SB TGAI/g in feed

Details on kinetic parameters:

Results of the study determined, in juvenile rainbow trout tissues, the parameters relevant to
assessing the potential for environmental biomagnification of the test material, Mortrace™
SB TGAI. These are:
Elimination rate constant (overall) 3.88 × 10-1 (days)-1
Growth-corrected elimination rate constant (kdepuration ) 3.41 × 10-1 (days)-1
Growth-corrected half-life (t1/2 ) 2.04 days
Assimilation efficiency (α) 7.05 × 10-2
Bioconcentration factor1 BCFLow estimate 8.42 × 102
Bioconcentration factor BCFHigh estimate 1.07 × 103
Biomagnification factor (BMF) 6.21 × 10-3
Lipid-normalized biomagnification factor BMFL 1.74 × 10-2

Metabolites:

not applicable

Results with reference substance (positive control):

HCB residues in tissue: The concentration of HCB on day 14 of uptake ranged from 9.93 to 16.6 μg/g (mean ±SD: 14.2 ±2.54 μg/g) in whole fish, 4.69 to 7.78 μg/g (mean ±SD: 6.21 ±1.34 μg/g) in fillet tissue, and 38.0 to 56.6 μg/g (mean ±SD: 49.0 ±7.69 μg/g) in viscera tissue. The measured HCB residues in whole fish, fillet, and viscera QC fortifications ranged from 105 to 117% of the nominal concentration. No residues of HCB were detected in the control tissues (i.e., whole fish, fillet, or viscera) above the MQL of 8.27 or 16.5 μg/g. A conservative estimate of the net assimilation efficiency (α) based on the mean measured HCB concentration in whole fish was 0.42, or 42%.

Details on results:

Range finding test:
Mortrace™ SB TGAI residues on day 10 of uptake ranged from 0.00786 to 0.0152 μg/g (mean ± SD: 0.0105 ± 0.00405 μg/g) in whole fish tissue, 0.00633 to 0.00898 μg/g (mean ± SD: 0.00803 ± 0.00148 μg/g) in fillet tissue, and 0.0329 to 0.0394 μg/g (mean ± SD: 0.0354 ± 0.00352 μg/g) in viscera tissue. HCB residues on day 10 of uptake ranged from 11.7 to 16.4 μg/g (mean ± SD: 13.7 ± 2.41 μg/g) in whole fish tissue, 5.92 to 9.39 μg/g (mean ± SD: 8.12 ± 1.91 μg/g) in fillet tissue, and 17.4 to 28.6 μg/g (mean ± SD: 24.1 ± 5.92 μg/g) in viscera tissue. No residues of Mortrace™ SB TGAI or HCB were detected in the control tissue above the MQL of 0.0210 μg/g for Mortrace™ SB TGAI or 0.187 μg/g for HCB. The measured Mortrace™ SB TGAI residues in whole fish and viscera QC
fortifications were 97 and 84%, respectively, of the nominal concentrations. The measured HCB residues in whole fish, fillet, and viscera QC fortifications ranged from 83 to 118% of the nominal concentrations.
The mean measured concentrations in feed offered during the uptake exposure were 1.86 μg Mortrace™ SB TGAI/g and 102 μg HCB/g when sampled three days following preparation. Recoveries of Mortrace™ SB TGAI from three grab samples of the feed, each analyzed in triplicate, ranged from 85 to 106% of the 2.0 μg/g nominal concentration when sampled three days following preparation and ranged from 79 to 88% for two grab samples analyzed in duplicate when sampled approximately three months following preparation. Recoveries of HCB in the same feed samples, analyzed at the same intervals, ranged from 94 to 113% of the 100 μg/g nominal concentration. Visual estimations of portion of offered diet consumed, performed approximately 30 to 60 minutes following offering of diet, indicated 80 to 90% of both diets (i.e., control and treated) was consumed at each offering during the uptake phase. No mortality or abnormalities occurred during the exposure.
Based on these range-finding test results, the feed dose rates evaluated did provide sufficient accumulation of Mortrace™ SB TGAI and HCB for analytical quantification and the rates did not elicit toxicity or lead to avoidance of the dosed feed. Results of the dosed feed analyses indicated the method used to amend Mortrace™ SB TGAI and HCB to the feed did facilitate homogenous distribution of the test and reference substances within the dosed feed as well as obtaining the target nominal concentration. The range-finding test results further supported the suitability of the analytical methods for recovering Mortrace™ SB TGAI and
HCB from feed and tissue.

Definitive test:
Feed: The mean measured concentrations in feed offered during the uptake exposure were 2.22 (±0.0656) μg Mortrace™ SB TGAI/g and 92.3 (±8.35) μg HCB/g when sampled one day following preparation. Recoveries of Mortrace™ SB TGAI from three grab samples of the feed, each analyzed in triplicate, ranged from 108 to 117% of the 2.0 μg/g nominal concentration when sampled one day following preparation and for HCB ranged from 82 to 108% of the 100 μg/g nominal concentration. The mean measured concentrations in feed sampled 22 days following preparation were 1.93 (±0.172) μg Mortrace™ SB TGAI/g and 116 (±1.54) μg HCB/g. Recoveries of Mortrace™ SB TGAI from three grab samples of the feed, each analyzed in triplicate, ranged from 79 to 109% of the 2.0 μg/g nominal concentration when sampled 22 days following preparation and for HCB ranged from 113 to 118% of the 100 μg/g nominal concentration. Visual estimations of portion of offered diet consumed, performed approximately 30 to 60 minutes following offering of diet, indicated 70 to 100% of both diets (i.e., control and treated) was consumed at each offering during the uptake phase, with the exception of the second feeding on day 10 of uptake. The visual estimate of the portion of offered diet consumed following the second feeding on day 10 of uptake indicated 30 to 50% of both diets (i.e., control and treated) was consumed. The lower percentage of consumed food at this observation point may have been the result of the diet being offered within four hours of the initial offering on day 10 of uptake.
Bilogical observations: Five control mortalities (four in replicate A and one in replicate B) and five treated mortalities (three in replicate A and two in replicate B) occurred during the uptake exposure. One mortality occurred in control replicate B during the depuration exposure. The mortalities did not appear to be related to the chemical exposure since the number of mortalities was similar in the control and treated treatments. All remaining fish were without abnormalities (i.e., normal) during the exposures.
Growth data: The control fish growth rate was 0.0464 g/day (90% confidence interval: 0.0381 and 0.0547 g/day). The treated fish growth rate was 0.0486 g/day (90% confidence interval: 0.0418 and 0.0554 g/day). The growth rate for pooled control and treated fish was 0.0475 g/day (90% confidence interval: 0.0422 and 0.0528 g/day). The growth rate for pooled control and treated fish was used as the overall fish growth rate (kgrowth) for all further calculations.
Mortrace SB TGAI residues in tissue: The concentration of Mortrace™ SB TGAI in treated whole fish tissue on day 14 of uptake ranged from 0.0184 to 0.0333 μg/g (mean ±SD: 0.0270 ±0.00621 μg/g). On day 1 of depuration, the concentration of Mortrace™ SB TGAI in treated whole fish tissue ranged from 0.00334 to 0.00752 μg/g (mean ±SD: 0.00556 ±0.00621 μg/g) and mean Mortrace™ SB TGAI residues continued to decline during the remainder of the depuration exposure to The concentration of Mortrace™ SB TGAI in treated fillet tissue on day 14 of uptake ranged from 0.00331 to 0.0154 μg/g (mean ±SD: 0.00810 ±0.00523 μg/g) (Table 10). On day 1 of depuration, the concentration of Mortrace™ SB TGAI in treated fillet tissue ranged from The concentration of Mortrace™ SB TGAI in treated viscera tissue on day 14 of uptake ranged from 0.0405 to 0.141 μg/g (mean ±SD: 0.0838 ±0.0365 μg/g) (Table 10). On day 1 of depuration, the concentration of Mortrace™ SB TGAI in treated viscera tissue ranged from 0.0414 to 0.0592 μg/g (mean ±SD: 0.0487 ±0.00746 μg/g) and on day 7 of depuration ranged from 0.0112 to 0.0295 μg/g (mean ±SD: 0.0227 ±0.00805 μg/g).
The measured Mortrace™ SB TGAI residues in whole fish QC fortifications ranged from 0.0102 to 0.0180 μg/g (mean ±SD: 0.0143 ±0.00253 μg/g), or 78 to 138% of the nominal concentration. The measured Mortrace™ SB TGAI residues in fillet QC fortifications ranged from 0.0100 to 0.0180 μg/g (mean ±SD: 0.0128 ±0.00295 μg/g), or 77 to 138% of the nominal concentration. The measured Mortrace™ SB TGAI residues in viscera QC fortifications ranged from 0.00977 to 0.0193 μg/g (mean ±SD: 0.0147 ±0.00345 μg/g), or 75 to 148% of the nominal concentration. No residues of Mortrace™ SB TGAI were detected in the control tissues (i.e., whole fish, fillet, or viscera) above the MQL of 0.00423 μg/g on days 0 and 14 of uptake or 0.00211 μg/g for the remainder of the exposure.
Feed and tissue lipid fractions: The fraction of lipids in the control feed ranged from 16 to 18% (mean ±SD: 17 ±1.0%) and ranged from 13 to 16% (mean ±SD: 14 ±1.5%) in the treated feed. The fraction of lipids in the control whole fish at day 0 of uptake ranged from 3.8 to 6.0% (mean ±SD: 5.0 ±1.1). The fraction of lipids in the control whole fish at day 0 of depuration ranged from 4.9 to 5.8% (mean ±SD: 5.0 ±0.47). The fraction of lipids in the treated whole fish at day 0 of depuration ranged from 5.9 to 7.0% (mean ±SD: 6.0 ±0.55). The mean lipid fraction ratio (i.e., lipid normalization factor), based on the mean treated feed lipid fraction and the mean control whole fish lipid fraction on day 0 of update, was 0.357.
Water quality: Water temperature during the exposure ranged from 13.5 to 14.7°C. The continuous temperature recorded with the data logger, demonstrated that the temperature of the test solutions remained within the range of 13.1 to 14.7°C. Dissolved oxygen concentrations ranged from 8.2 to 11.9 mg/L and ranged from 83 to 120% saturation. The pH ranged from 7.7 to 8.4 in all solutions. The dilution water total hardness and alkalinity ranged from 140 to 154 mg CaCO3/L and 144 to 158 mg CaCO3/L, respectively. The conductivity of the dilution water ranged from and 348 to 370 μS.

Reported statistics:

Standard statistical methods were employed

Mortrace™ SB TGAI residues in whole fish tissues fortified at 0.0206 μg/g ranged from 80 to 97% of the nominal concentration and ranged from 76 to 100% in whole fish tissues fortified at 0.499 μg/g. HCB residues in whole fish tissues fortified at 10.1 μg/g ranged from 113 to 121% of nominal. No residues of Mortrace™ SB TGAI were detected in the control whole fish tissues above the MQL of 0.0114 μg/g. These results showed the analytical method was suitable for recovering Mortrace™ SB TGAI and HCB from fish tissue. Mortrace™ SB TGAI residues in feed fortified at 2.14 μg/g ranged from 110 to 120% of the nominal concentration. HCB residues in feed fortified at 101 μg/g ranged from 99 to 112% of nominal. With the exception of one of the three samples, no residues of Mortrace™ SB TGAI were detected in the control feed above the MQL of 0.103 μg/g. One control sampled contained 0.105 μg/g of Mortrace™ SB TGAI. No residues of HCB were detected in the control feed above the MQL of 0.336 μg/g. These results showed the analytical method was suitable for recovering Mortrace™ SB TGAI and HCB from the feed.

Validity criteria fulfilled:

yes

Conclusions:

Results of the study determined, in juvenile rainbow trout tissues, the parameters relevant to assessing the potential for environmental biomagnification of the test material, Mortrace™ SB TGAI. These are:
Elimination rate constant (overall) 3.88 × 10-1 (days)-1
Growth-corrected elimination rate constant (kdepuration ) 3.41 × 10-1 (days)-1
Growth-corrected half-life (t1/2 ) 2.04 days
Assimilation efficiency (α) 7.05 × 10-2
Bioconcentration factor1 BCFLow estimate 8.42 × 102
Bioconcentration factor BCFHigh estimate 1.07 × 103
Biomagnification factor (BMF) 6.21 × 10-3
Lipid-normalized biomagnification factor BMFL 1.74 × 10-2
Of the above parameters, the lipid-normalized BMF is the most relevant to estimating the potential for Mortrace™ SB TGAI to biomagnify in the environment. The result supports the conclusion that Mortrace™ SB TGAI will not biomagnify in the environment.
(Bioconcentration Factors derived from Biomagnification Factors are highly conservative because to determine the BCF it is assumed that the test substance is completely soluble in the water phase. For Mortrace™ SB TGAI this assumption is not valid because Mortrace™ SB TGAI is not water soluble to any appreciable degree. Therefore, the calculated BCF values are highly conservative estimates and possibly inappropriate for use in standard environmental fate models).

Executive summary:

A test was conducted to estimate the potential for accumulation of Mortrace™SB TGAI within juvenile rainbow trout,Oncorhynchus mykiss, via a dietary exposure. The objective of this study was to determine the half-life (t½, from the elimination rate constant, kdepuration), the assimilation efficiency (α), the biomagnification factor (BMF), the lipid-normalized biomagnification factor (BMFL), and bioconcentration factor (BCF) for Mortrace™SB TGAI. One group of juvenile rainbow trout was offered a treated diet consisting of 2µg Mortrace™SB TGAI (Lot No. ZA78743-5-2S; 98% active ingredient; TD No. 05-014) (i.e., test substance) and 100 µg hexachlorobenzene (i.e., reference substance) per gram of feed. Hexaclorobenzene was included in the treated diet as a benchmark substance to evaluate assimilation efficiency of the treated diet since it is known to readily bioaccumulate and not depurate significantly. A second group of juvenile rainbow trout was offered an acetone-dosed control diet. Beginning on day 0 and ending on day 13 of the uptake phase, control fish were offered the acetone dosed control diet and the treated fish were offered the treated diet at a level of approximately 1.5% of body weight twice each day (i.e., total of 3% of body weight each day). The depuration phase was initiated immediately following the sampling of fish on day 14 of uptake. During depuration, fish in the control and treated test chambers were offered a non-treated, non-acetone dosed, diet. The exposure method was developed as an alternative for those methods outlined in the OECD Guideline 305 and U.S. EPA Ecological Effects Testing Guideline OPPTS 850.1730, to address bioaccumulation testing of a poorly water soluble test substance.

 

Survival was monitored daily by visually inspecting each test chamber, and any behavioral or physical changes were recorded, including abnormalities. Five control mortalities and five treated mortalities occurred during the uptake exposure. One mortality occurred in the control during the depuration exposure. The mortalities did not appear to be related to the chemical exposure since the number of mortalities was similar in the control and treated treatments. All remaining fish were without abnormalities (i.e., normal) during the exposures.

 

Visual estimations of portion of offered diet consumed indicated the juvenile rainbow trout did not avoid consuming the Mortrace™SB TGAI and hexachlorobenzene treated diet since 70 to 100% of both the control and treated diets was consumed at each offering, with the exception of the second feeding on day 10 of uptake. The visual estimate of the portion of offered diet consumed following the second feeding on day 10 of uptake indicated 30 to 50% of both diets (i.e., control and treated) was consumed. The lower percentage of consumed food at this observation point may have been the result of the diet being offered within four hours of the initial offering on day 10 of uptake.

 

Fish were sampled on days 0 and 14 of uptake and days 1, 2, 4, 7, 14 and 21 of depuration. Whole fish samples collected at each sample point were analyzed for Mortrace™SB TGAI residues. Fillet tissue (i.e., tissue without internal organs) and viscera tissue (i.e., internal organs) collected on days 0 and 14 of uptake and days 1 and 7 of depuration were analyzed for Mortrace™SB TGAI residues. Hexachlorobenzene residues were also measured in all tissue (i.e., whole fish, fillet and viscera) collected on days 0 and 14 of uptake to evaluate the assimilation efficiency of the treated diet and was not measured thereafter through depuration. Water quality characteristics of temperature, dissolved oxygen concentration, and pH were measured at test initiation, twice per week throughout the test, and at test termination, and remained within acceptable limits throughout the exposure. All test solutions appeared clear and colorless throughout the exposure with no visible particulates, surface film, undissolved test substance, or precipitate.

 

The measured concentrations of HCB in whole fish (14.2µg/g), fillet (6.21µg/g), and viscera (49.0µg/g) tissues, along with visual estimates of treated diet consumed, indicate the juvenile rainbow trout consumed the diet offered during the 14 days of uptake. A conservative estimate of the net assimilation efficiency based on the mean measured HCB concentration in whole fish was 0.41, or 41%. Survival and growth of the treated fish were similar to controls, indicating the treated diet had no effect on juvenile rainbow trout survival and growth. The growth rate for pooled control and treated fish was 0.0475 g/day and was used as the overall fish growth rate (kgrowth) for all further calculations.

 

All analytical methods in this study were validated and the study was conducted in compliance with GLP with the exception that the purity of hexachlorobenzene marker was not determined under GLP. The control mortality was <10% at the end of the test, the concentration of dissolved oxygen remained above 60% saturation, and there were no detectable residues above the minimum quantifiable limit of the test substance found in the untreated food or control fish tissues. The objective of the study was achieved; therefore, the study was deemed valid.

 

Results of the study determined, in juvenile rainbow trout tissues, the parameters relevant to

assessing the potential for environmental biomagnification of the test material, Mortrace™

SB TGAI. These are:

Elimination rate constant (overall) 3.88 × 10-1(days)-1

Growth-corrected elimination rate constant (kdepuration) 3.41 × 10-1(days)-1

Growth-corrected half-life (t1/2) 2.04 days

Assimilation efficiency (α) 7.05 × 10-2

Bioconcentration factor1BCFLow estimate8.42 × 102

Bioconcentration factor BCFHigh estimate1.07 × 103

Biomagnification factor (BMF) 6.21 × 10-3

Lipid-normalized biomagnification factor BMFL1.74 × 10-2

(Bioconcentration Factors derived from Biomagnification Factors are highly conservative because to determine the BCF it is assumed that the test substance is completely soluble in the water phase. For Mortrace™SB TGAI this assumption is not valid because Mortrace™SB TGAI is not water soluble to any appreciable degree. Therefore, the calculated BCF values are highly conservative estimates and possibly inappropriate for use in standard environmental fate models.)

Description of key information

A dietary bioaccumulation study was conducted due to poor water solubility of the test substance.  The estimated BCF was 842 - 1070.  This BCF value is likely over-conservative  because to determine the BCF from a dietary study, it is assumed that the test substance is completely soluble in the water phase, which is not the case for Mortrace SB.

Key value for chemical safety assessment

BCF (aquatic species):

1 070 dimensionless

Additional information

Juvenile rainbow trout were administered Mortrace SB (2 ug/g) in the diet for 14 days followed by a 21 -day depuration phase. Results of the study determined, in juvenile rainbow trout tissues, the parameters relevant to assessing the potential for environmental biomagnification of the test material, Mortrace™ SB TGAI. These are:

Elimination rate constant (overall): 3.88 × 10^-1(days)-1

Growth-corrected elimination rate constant (kdepuration): 3.41 × 10^-1(days)-1

Growth-corrected half-life (t1/2): 2.04 days

Assimilation efficiency (α): 7.05 × 10^-2

Bioconcentration factor1BCFLow estimate: 8.42 × 10²

Bioconcentration factor BCFHigh estimate: 1.07 × 10³

Biomagnification factor (BMF): 6.21 × 10^-3

Lipid-normalized biomagnification factor BMFL: 1.74 × 10 ^-2

Bioconcentration Factors derived from Biomagnification Factors are highly conservative because to determine the BCF it is assumed that the test substance is completely soluble in the water phase. For Mortrace™SB TGAI this assumption is not valid because Mortrace™SB TGAI is not water soluble to any appreciable degree. Therefore, the calculated BCF values are highly conservative estimates and possibly inappropriate for use in standard environmental fate models.