Phenol, paraalkylation products with C12-rich branched olefins derived from propene oligomerisation, ethane-1,2-diol, carbonate, sulfurized, calcium salts, overbased, reacted with alkylene carbonate, including distillates (petroleum), heavy paraffinic C10-C50

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:

5-December 2014 to 15-October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Test Guideline Adopted: 2 October 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1730 (Fish Bioconcentration Test)

Version / remarks:

EPA 712–C–96–129; April 1996

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Reference material: EXP1200078, Batch/Lot No.: E00275-350; very dark brown (almost black), viscous liquid.

Radiolabeled test substance: a liquid, was identified as [14C]EXP1200078 CFQ42196; specific radioactivity of 1.77 µCi/mg.

Radiolabelling:

yes

Details on sampling:

Analytical Sampling
Water samples were collected and analyzed for total radioactivity. Water samples were collected from the Tween® control and each of the 5.0 and 50 µg/L
treatment groups, 5 and 1 days prior to the start of the exposure period to confirm the concentrations after conditioning the diluter system. In the Tween®
control and both treatment groups, water samples were collected on days 0 (hours 0 and 4), 1, 3, 7, 14, 21, 28, 35, 42, and 49 of the uptake phase, and days 1, 3, 7, 10, 15, 28, and 61 of the depuration phase for the analysis of 14C-labeled test substance. Additional water samples were collected on days 16, 17
and 21 of the uptake phase after a diluter malfunction occurred. On each sampling day, typically one water sample was analyzed from the control test
chamber and two from each treatment group test chambers. All water samples for the analysis of total radioactivity were collected in 20-mL glass LSC vials from mid-depth in the test chamber using a glass volumetric pipette.

Water samples were processed immediately for analysis. In addition, two 1 L water samples were collected after steady state was confirmed. These samples were collected on Day 50 of the uptake phase from the control and both treatment groups. Water samples were then stored frozen for possible analysis of metabolites. Additional water samples were collected from the high treatment group on Day 42 of the uptake phase for fractionation.

Collection of Tissue Samples
Tissue samples were collected from the Tween® control and both treatment groups on uptake days 0 (hour 4), 1, 3, 7, 14, 21, 28, 35, 42, and 49 and on depuration days 1, 3, 7, 10, 15, 28, and 61. At each tissue sampling interval, a sufficient number of fish were collected to provide at least four replicate samples of Tween® control fish and four replicate samples from each treatment group, except on Day 61 of the depuration phase in the low treatment group where only 3 fish remained for analysis. Fish were impartially removed from the test chambers, rinsed with dilution water, blotted dry and euthanized by making an incision from just posterior to the base of the pectoral fin dorsally through the spinal cord. The fish were measured for total length and wet weight within approximately 15 minutes of collection. Each fish was then dissected and divided into edible tissues and non-edible tissues. The head, fins and viscera were removed from the body and were considered to be non edible tissue. The remaining tissue was considered the edible tissue. Edible and non-edible tissue samples were transferred to pre-weighed glass vials and weighed. All tissue samples were processed immediately or stored frozen.

To evaluate the presence of undigested food in the gut tract, additional fish were collected at the end of the uptake phase from the control and treatment groups. After sampling, the gut tract was removed from each fish. The separated gut tract and corresponding whole fish samples were transferred to pre weighed glass vials. All tissues were processed immediately or stored frozen.


Lipid Sampling
Twenty-seven additional fish were collected from the control and each treatment group to determine lipid content (nine from the control and each treatment group). Of these fish sampled, one fish was analyzed for lipid content from the control and each treatment group at each interval and the remaining were held as back-ups. Fish for lipid analysis were sampled on Day 0 of uptake, after confirmation of steady state (Day 50 uptake) and at the end of depuration (Day 61 depuration). All fish collected for lipid content were stored frozen until analysis.

Analytical Method for the 14C Analysis of the Test Substance
The analysis of the test samples were based upon methodology developed by Wildlife International. Freshwater and fish tissue samples were collected from a test designed to determine the bioconcentration potential of 14C test substance in bluegill. The analyses of these samples were performed at Wildlife International using liquid scintillation counting (LSC).

The concentrations of the test substance in freshwater and fish tissue were verified by liquid scintillation counting (LSC). For aqueous samples, 10.0 mL of Ultima Gold XR scintillation cocktail was added to scintillation vials. Tissue samples were weighed and combusted in a Packard sample oxidizer prior to LSC analysis. A Perkin Elmer Packard TriCarb 2910 liquid scintillation analyzer was used to determine disintegrations per minute (dpm). Instrument parameters for LSC are
presented in Appendix 6.1 and a method flowchart for water and tissue is presented in Appendix 6.2. The calculations for sample quantitation and limits of quantitation for both tissue and water are presented in Appendix 6.3.


Lipid Analyses
HPLC-grade water (10 mL) and a steel grinding ball were added to each tissue sample in polycarbonate Geno/Grinder® vials. Samples were placed on Geno/Grinder® and homogenized for 5 minutes at 1200 rpm. Each homogenate was transferred to a 250 mL separatory funnel containing 25 mL of chloroform and 50 mL of methanol. A strong magnet held on the bottom of the vial was used to prevent the transfer of the steel ball to the separatory funnel. A second 10 mL aliquot of HPLC-grade water was added to each sample vial. The samples were capped and shaken, then the rinse was combined into each samples’ respective separatory funnel. A strong magnet held on the bottom of the vial was used to prevent the transfer of the steel ball to the separatory funnel. The funnels were then shaken with venting for approximately one minute, and 50 mL of chloroform followed by 50-mL of saturated sodium chloride in HPLC-grade water was added to each separatory funnel. Funnels were swirled briefly with venting and the phases were allowed to separate overnight. For each sample, the chloroform layer was drained through a powder funnel packed with glass wool and anhydrous sodium sulfate into a 250 mL roundbottom flask. An additional 50-mL aliquot of chloroform was added to each separatory funnel and the extraction and draining procedures were repeated allowing approximately 60 minutes for phase separation. The extracts were rotary-evaporated to near dryness in a waterbath maintained at approximately 40 C. Each sample was then transferred to a pre-weighed (without cap), labeled scintillation vial. Each 250-mL roundbottom flask was rinsed with a small volume of chloroform and the rinse was transferred to its respective scintillation vial. The remaining solvent in each vial was evaporated under a gentle stream of nitrogen using a multivap. Each vial was reweighed and the weight recorded. The lipid content was calculated from the difference in the vial tare weight and the vial weight containing the lipid. Percent lipids were calculated from the ratio of lipid content to total tissue sample weight. A method flowchart is presented in Appendix 6.4 and lipid content determinations are presented in Appendix 6.5.

Vehicle:

yes

Remarks:

The concentration of Tween®20 in the control and all treatment groups was 0.1 mL/L.

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

Preparation of Test Solutions
Five (14C) primary stock solutions were prepared during the study at a target nominal concentration of 20 mg/mL. The radioactivity of the stocks prepared was confirmed prior to use in the study by analyzing three samples of the stock solution by liquid scintillation counting (LSC).

The primary stock solutions of 14C-test substance were prepared by mixing a calculated amount (0.4000 g) of test substance into 2 mL of the solubilizing agent Tween20® in a calibrated beaker. The calibrated beaker was brought to volume (20 mL) with Tween20 adjusted reverse osmosis (RO) water and stirred for 15 minutes. All 14C-test substance primary stock solutions were opaque and dark brown in color.

The dispensing stocks were prepared at nominal concentrations of 0.050 and 0.50 mg/mL. The dispensing stocks were prepared by mixing the appropriate volumes of radiolabeled test substance primary stock solutions and bringing the stocks to volume with Tween20 adjusted RO water. The radioactivity of the dispensing stock solutions was approximately 196,470 and 1,964,700 dpm/mL in the low and high dispensing stocks, respectively. The final nominal radioactivity in each of the aqueous exposure solutions were 19.6 and 196 dpm/mL in the low and high treatment groups, respectively. Dispensing stocks were prepared in 100 to 500 mL volumes and mixed by inversion. The Tween20 control stock appeared clear and light yellow in color, the 0.050 mg/mL stock appeared clear and slightly brown in color and the 0.50 mg/mL stock was slightly translucent tan in color with a brown precipitate. The final specific activity in the low (5 µg/L) and high (50 µg/L) exposure levels was 3929 dpm/µg.

Stock solutions were stored refrigerated in glass amber bottles, and aliquots of each stock were placed in the syringe daily during the uptake phase of the study. The dispensing stocks were pumped into the diluter mixing chambers assigned to the treatment groups at a target rate of 35.0 µL/min and were mixed with well water in the mixing chambers, delivered at a target rate of 350 mL/min, to achieve the nominal test concentrations of 5 and 50 µg/L. The Tween® control was prepared by delivering Tween20 adjusted RO (0.1 mL Tween / 1 mL RO water) water to the mixing chamber for the Tween® control. The concentration of Tween®20 in the control and all treatment groups was 0.1 mL/L. Dilution water was delivered at a target rate of 197 mL/min during the depuration phase. Flows to the test system were initiated 6 days prior to initiation of the uptake phase. The test solutions in the mixing chambers and test chambers appeared clear and colorless during both the uptake and depuration phases. There was a white foam throughout the solution in the mixing chambers of the Tween control and high treatment group at test initiation, and in the control and both treatment groups at end of uptake. There was also a slight of amount of white foam at the water surface of the high treatment group at test initiation.

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

Test Organism
The bluegill, (Lepomis macrochirus), was selected as the test species for this study. This species is representative of an important group of aquatic vertebrates and was selected for use in the test based upon past history of use in the laboratory. It is also a recommended test species in the cited test guidelines for performing fish bioaccumulation studies. Bluegill used in the test were obtained from Osage Catfisheries, Inc., Osage Beach, Missouri and hatched on April 17, 2014. Identification of the species was verified by the supplier. The recommended length for fish used in the study was 5.0 ± 2.0 cm. The average length and weight of fish sampled on Day 0 of the uptake phase was 5.4 ± 0.42 cm (4.5 – 6.0 cm) and 2.02 ± 0.441 g (1.26 – 2.69 g), respectively.

The fish were held for at least 14 days prior to the test in water from the same source and at approximately the same temperature as used during the test. During the 2 week period immediately preceding the test, water temperatures ranged from 21.5 to 22.8¿C, measured with a hand-held liquid in-glass thermometer. The pH of the water ranged from 8.1 to 8.4, measured with a Thermo Orion Benchtop 4 Star Plus pH/ISE meter. Dissolved oxygen ranged from 8.3 to 8.7 mg/L (>96% of saturation), measured with a Thermo Orion Benchtop 3 Star Plus dissolved oxygen meter. All fish in the culture during this time appeared normal. At test initiation, the bluegill were collected from the holding tanks and impartially distributed two to three at a time to the test chambers until the Tween® control and each treatment level test chamber contained 90 fish.

Loading
Biomass loading rates were calculated using the wet weights of the fish collected on Day 0 since this was considered to be representative of the biomass in test chambers during the test. All fish used in the test were from the same source and year class. Loading was defined as the total wet weight of fish per liter of test water that passed through the test chamber in 24 hours, and was determined to be 0.36 g fish/L/day. Instantaneous loading (the total wet weight of fish per liter of water in the tank) was 2.3 g fish/L.

Feeding
During the holding period and the test, fish were fed at least once daily a commercially prepared diet of Sera Vipan supplied by Sera, North America of Montgomeryville, Pennsylvania. The fish were not fed on the last day of the test so that the gut contents could be purged. Uneaten food and feces were siphoned daily from the test chambers shortly after feeding. The diet was fed at a rate of 2% of fish body weight. To ensure that the feeding rate per fish remained constant, the amount of food supplied to each test chamber was adjusted at least weekly based on weight of the fish. Specifications for acceptable levels of contaminants in fish diets has not been established. However, there were no known levels of contaminants reasonably expected to be present in the diet that were considered to interfere with the purpose or conduct of the test. The results of periodic analyses performed to measure the concentrations of selected contaminants in the
feed used by Wildlife International are presented in Appendix 3.

Dilution Water
The water used for holding and testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International site. The well water is characterized as moderately-hard water. The well water was passed through a sand filter to remove particles greater than approximately 25 ¿m, and pumped into a 37,800-L storage tank and aerated with spray nozzles. Prior to use, the water was filtered to 0.45 ¿m to remove fine particles and was passed through an ultraviolet (UV) sterilizer. The results of periodic analyses performed to measure the concentrations of selected contaminants in well water used by Wildlife International are presented in Appendix 4. The specific conductance, hardness, alkalinity, pH and total organic carbon (TOC) of the well water during the four-week period immediately preceding the test are presented in Appendix 5.

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason:

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

49 d

Total depuration duration:

61 d

Hardness:

132 - 144 mg/L as CaCO3

Test temperature:

21.2 to 22.2 C

pH:

7.8 to 8.3

Dissolved oxygen:

7.2 to 8.7 µg/L

TOC:

Mean Measured Test Concentration (µg/L)
Uptake Phase: Tween control, 4.8, 48, dilution water: 10.19, 8.699, 8.353, 4.697 (µg/L)

Depuration Phase: Tween control, 4.8, 48, dilution water: 2.484, 2.181, 2.377, 1.841 (µg/L)

Salinity:

Not measured

Conductivity:

297 to 390 µS/cm

Details on test conditions:

Loading
Biomass loading rates were calculated using the wet weights of the fish collected on Day 0 since this was considered to be representative of the biomass in test chambers during the test. All fish used in the test were from the same source and year class. Loading was defined as the total wet weight of fish per liter of test water that passed through the test chamber in 24 hours, and was determined to be 0.36 g fish/L/day. Instantaneous loading (the total wet weight of fish per liter of water in the tank) was 2.3 g fish/L.

Feeding
During the holding period and the test, fish were fed at least once daily a commercially prepared diet of Sera Vipan supplied by Sera, North America of Montgomeryville, Pennsylvania. The fish were not fed on the last day of the test so that the gut contents could be purged. Uneaten food and feces were siphoned daily from the test chambers shortly after feeding. The diet was fed at a rate of 2% of fish body weight. To ensure that the feeding rate per fish remained constant, the amount of food supplied to each test chamber was adjusted at least weekly based on weight of the fish. Specifications for acceptable levels of contaminants in fish diets has not been established. However, there were no known levels of contaminants reasonably expected to be present in the diet that were considered to interfere with the purpose or conduct of the test. The results of periodic analyses performed to measure the concentrations of selected contaminants in the feed used by Wildlife International are presented in Appendix 3.

Dilution Water
The water used for holding and testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International site. The well water is characterized as moderately hard water. The well water was passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800-L storage tank and aerated with spray nozzles. Prior to use, the water was filtered to 0.45 µm to remove fine particles and was passed through an ultraviolet (UV) sterilizer. The results of periodic analyses performed to measure the concentrations of selected contaminants in well water used by Wildlife International are presented in Appendix 4. The specific conductance, hardness, alkalinity, pH and total organic carbon (TOC) of the well water during the four week period immediately preceding the test are presented in Appendix 5.

Test Apparatus
The bioconcentration test was conducted using an exposure system consisting of a continuous flow diluter to deliver each concentration of the test substance and a Tween® control to test chambers. A syringe pump (Harvard Apparatus, Holliston, Massachusetts) was used to deliver volumes of test substance stock solutions and the Tween® control to mixing chambers indiscriminately assigned to each treatment and the control. The stock solutions or Tween® were diluted with well water in the mixing chambers in order to obtain the desired test concentrations prior to delivery to the test chambers. The flow of dilution water into each mixing chamber was controlled using rotameters, and was adjusted to provide approximately 6 exchange additions of test solution in each test chamber per day. The general operation of the diluter was checked at least two times a day during the test, and at least once at the beginning and end of the test. The pumps used to deliver stock solutions and Tween® to the mixing chambers were calibrated prior to the test. The rotameters used to control the flow of dilution water to the mixing chambers, were calibrated prior to the test and verified weekly throughout the uptake phase.

Test chambers were positioned in a temperature-controlled water bath designed to maintain the target temperature throughout the test period. Test chambers used during the uptake phase were 127-L Teflon®-lined stainless steel aquaria filled with 80 L of test solution. The depth of the test water in one representative test chamber during uptake was approximately 17.5 cm. On day 50 of uptake, the fish were transferred to a clean set of 54-L stainless steel aquaria filled with 45 L of dilution water (well water) for 61 days of depuration. The depth of the test water in one representative test chamber during depuration was approximately 23.9 cm. Two separate water baths of the same type were used for the uptake and depuration phases. Test chambers were siphoned daily during the test to remove excess feed, fecal matter, algae and bacterial growth. Test chambers were identified by the project number and test concentration.

Non-GLP Rangefinding Study
A preliminary 96-hour static renewal range-finder was conducted at nominal concentrations of 0.081, 0.27, 0.90, 3.0 and 10 mg/L. This range-finder was conducted to identify an acute LC50 value to aid in setting test concentrations for the definitive study. Based on the non-GLP rangefinder, the 96-hour LC50 was >10 mg/L (Appendix 17).

Nominal and measured concentrations:

Measured concentrations via total detected isotope.
Low-dose: 5 ug/L (nominal); 4.8 ug/L (measured)
High-dose : 50 ug/L (nominal); 48 ug/L (measured)

Reference substance (positive control):

no

Details on estimation of bioconcentration:

The detailed equations to estimate bioconcentration are described in Appendix 7 which is a separate attachment.

Lipid content:

>= 2.55 %

Time point:

start of exposure

Lipid content:

>= 3.17 %

Time point:

other: Day 50 of uptake

Lipid content:

>= 4.11 %

Time point:

other: Day 61 of depuration

Key result

Conc. / dose:

4.8 µg/L

Temp.:

>= 21.2 - <= 22.2 °C

pH:

8.1

Type:

BCF

Value:

37 dimensionless

Basis:

edible fraction

Time of plateau:

49 d

Calculation basis:

steady state

Remarks:

Concentration in the water is expressed as equivalents of test substance based on total radioactivity and specific activity.

Remarks on result:

other: Based on total radioactivity tissue concentration and expressed as equivalents of test substance using specific activity.

Remarks:

Range of pH is 7.8 to 8.3 during uptake and depuration; mid-point of 8.1 inserted above.

Key result

Conc. / dose:

4.8 µg/L

Temp.:

>= 21.2 - <= 22.2 °C

pH:

8.1

Type:

BCF

Value:

233 dimensionless

Basis:

non-edible fraction

Remarks:

Concentration in the water is expressed as equivalents of test substance based on total radioactivity and specific activity.

Time of plateau:

49 d

Calculation basis:

steady state

Remarks:

Concentration in the water is expressed as equivalents of test substance based on total radioactivity and specific activity.

Remarks on result:

other: Based on total radioactivity tissue concentration and expressed as equivalents of test substance using specific activity.

Remarks:

Range of pH is 7.8 to 8.3 during uptake and depuration; mid-point of 8.1 inserted above.

Key result

Conc. / dose:

4.8 µg/L

Temp.:

>= 21.2 - <= 22.2 °C

pH:

8.1

Type:

BCF

Value:

135 dimensionless

Basis:

whole body w.w.

Time of plateau:

49 d

Calculation basis:

steady state

Remarks:

Concentration in the water is expressed as equivalents of test substance based on total radioactivity and specific activity.

Remarks on result:

other: Based on total radioactivity tissue concentration and expressed as equivalents of test substance using specific activity.

Remarks:

Range of pH is 7.8 to 8.3 during uptake and depuration; mid-point of 8.1 inserted above.

Key result

Conc. / dose:

48 µg/L

Temp.:

>= 21.2 - <= 22.2 °C

pH:

8.1

Type:

BCF

Value:

44 dimensionless

Basis:

edible fraction

Time of plateau:

49 d

Calculation basis:

steady state

Remarks:

Concentration in the water is expressed as equivalents of test substance based on total radioactivity and specific activity.

Remarks on result:

other: Based on total radioactivity tissue concentration and expressed as equivalents of test substance using specific activity.

Remarks:

Range of pH is 7.8 to 8.3 during uptake and depuration; mid-point of 8.1 inserted above.

Key result

Conc. / dose:

48 µg/L

Temp.:

>= 21.2 - <= 22.2 °C

pH:

8.1

Type:

BCF

Value:

254 dimensionless

Basis:

non-edible fraction

Time of plateau:

49 d

Calculation basis:

steady state

Remarks:

Concentration in the water is expressed as equivalents of test substance based on total radioactivity and specific activity

Remarks on result:

other: Based on total radioactivity tissue concentration and expressed as equivalents of test substance using specific activity.

Remarks:

Range of pH is 7.8 to 8.3 during uptake and depuration; mid-point of 8.1 inserted above.

Key result

Conc. / dose:

48 µg/L

Temp.:

>= 21.2 - <= 22.2 °C

pH:

8.1

Type:

BCF

Value:

146 dimensionless

Basis:

whole body w.w.

Time of plateau:

49 d

Calculation basis:

steady state

Remarks:

Concentration in the water is expressed as equivalents of test substance based on total radioactivity and specific activity.

Remarks on result:

other: Based on total radioactivity tissue concentration and expressed as equivalents of test substance using specific activity.

Remarks:

Range of pH is 7.8 to 8.3 during uptake and depuration; mid-point of 8.1 inserted above.

Key result

Conc. / dose:

ca. 5 µg/L

Temp.:

>= 21.2 - <= 22.2 °C

pH:

8.1

Type:

BCF

Value:

43 dimensionless

Basis:

edible fraction

Remarks:

Concentration in the water is expressed as equivalents of test substance based on total radioactivity and specific activity.

Time of plateau:

64.8 d

Calculation basis:

kinetic

Remarks:

Calculated using methods outlined in OPPTS guideline 850.1730.

Remarks on result:

other: Time to plateau - days to reach 95% per methods outlined in OPPTS guideline 850.1730.

Remarks:

Range of pH is 7.8 to 8.3 during uptake and depuration; mid-point of 8.1 inserted above.

Key result

Conc. / dose:

ca. 5 µg/L

Temp.:

>= 21.2 - <= 22.2 °C

pH:

8.1

Type:

BCF

Value:

364 dimensionless

Basis:

non-edible fraction

Remarks:

Concentration in the water is expressed as equivalents of test substance based on total radioactivity and specific activity.

Time of plateau:

68.8 d

Calculation basis:

kinetic

Remarks:

Calculated using methods outlined in OPPTS guideline 850.1730.

Remarks on result:

other: Time to plateau - days to reach 95% per methods outlined in OPPTS guideline 850.1730.

Remarks:

Range of pH is 7.8 to 8.3 during uptake and depuration; mid-point of 8.1 inserted above.

Key result

Conc. / dose:

>= 5 µg/L

Temp.:

>= 21.2 - <= 22.2 °C

pH:

8.1

Type:

BCF

Value:

204 dimensionless

Basis:

whole body w.w.

Remarks:

Concentration in the water is expressed as equivalents of test substance based on total radioactivity and specific activity.

Time of plateau:

67.4 d

Calculation basis:

kinetic

Remarks:

Time of plateau - Days to reach 95% Calculated using methods outlined in OPPTS guideline 850.1730.

Remarks on result:

other: Time to plateau - days to reach 95% per methods outlined in OPPTS guideline 850.1730.

Remarks:

Range of pH is 7.8 to 8.3 during uptake and depuration; mid-point of 8.1 inserted above.

Key result

Conc. / dose:

ca. 50 µg/L

Temp.:

>= 21.2 - <= 22.2 °C

pH:

8.1

Type:

BCF

Value:

55 dimensionless

Basis:

edible fraction

Remarks:

Concentration in the water is expressed as equivalents of test substance based on total radioactivity and specific activity.

Time of plateau:

79.6 d

Calculation basis:

kinetic

Remarks:

Calculated using methods outlined in OPPTS guideline 850.1730.

Remarks on result:

other: Time to plateau - days to reach 95% per methods outlined in OPPTS guideline 850.1730.

Remarks:

Range of pH is 7.8 to 8.3 during uptake and depuration; mid-point of 8.1 inserted above.

Key result

Conc. / dose:

ca. 50 µg/L

Temp.:

>= 21.2 - <= 22.2 °C

pH:

8.1

Type:

BCF

Value:

379 dimensionless

Basis:

non-edible fraction

Remarks:

Concentration in the water is expressed as equivalents of test substance based on total radioactivity and specific activity.

Time of plateau:

67.3 d

Calculation basis:

kinetic

Remarks:

Calculated using methods outlined in OPPTS guideline 850.1730.

Remarks on result:

other: Time to plateau - days to reach 95% per methods outlined in OPPTS guideline 850.1730.

Remarks:

Range of pH is 7.8 to 8.3 during uptake and depuration; mid-point of 8.1 inserted above.

Key result

Conc. / dose:

ca. 50 µg/L

Temp.:

>= 21.2 - <= 22.2 °C

pH:

8.1

Type:

BCF

Value:

223 dimensionless

Basis:

whole body w.w.

Remarks:

Concentration in the water is expressed as equivalents of test substance based on total radioactivity and specific activity.

Time of plateau:

68.5 d

Calculation basis:

kinetic

Remarks:

Calculated using methods outlined in OPPTS guideline 850.1730.

Remarks on result:

other: Time to plateau - days to reach 95% per methods outlined in OPPTS guideline 850.1730.

Remarks:

Range of pH is 7.8 to 8.3 during uptake and depuration; mid-point of 8.1 inserted above.

Key result

Elimination:

yes

Parameter:

other: DT95

Remarks:

Calculated using methods outlined in OPPTS guideline 850.1730.

Depuration time (DT):

64.8 d

Remarks on result:

other: Edible tissue

Remarks:

5 ug/L water - Nominal

Key result

Elimination:

yes

Parameter:

other: DT95

Remarks:

Calculated using methods outlined in OPPTS guideline 850.1730.

Depuration time (DT):

68.8 d

Remarks on result:

other: Non-edible tissue

Remarks:

5 ug/L water - Nominal

Key result

Elimination:

yes

Parameter:

other: DT95

Remarks:

Calculated using methods outlined in OPPTS guideline 850.1730.

Depuration time (DT):

67.4 d

Remarks on result:

other: Whole Fish w.w.

Remarks:

5 ug/L water - nominal

Key result

Elimination:

yes

Parameter:

other: DT95

Remarks:

Calculated using methods outlined in OPPTS guideline 850.1730.

Depuration time (DT):

79.6 d

Remarks on result:

other: Edible tissue

Remarks:

50 ug/L water - nominal

Key result

Elimination:

yes

Parameter:

DT50

Remarks:

Calculated using methods outlined in OPPTS guideline 850.1730.

Depuration time (DT):

18.38 d

Remarks on result:

other: Non-edible tissue

Remarks:

50 ug/L water – nominal

Key result

Elimination:

yes

Parameter:

other: DT95

Remarks:

Calculated using methods outlined in OPPTS guideline 850.1730.

Depuration time (DT):

68.5 d

Remarks on result:

other: Whole fish w.w.

Remarks:

50 ug/L water – nominal

Key result

Rate constant:

overall uptake rate constant (L kg-1 d-1)

Remarks:

SAS Institute, Inc. 1999 - 2001. The SAS System for Windows. Version 8.2. Cary, North Carolina.

Value:

2

Remarks on result:

other: Edible tissue

Remarks:

5 ug/L nominal test article in water

Key result

Rate constant:

overall uptake rate constant (L kg-1 d-1)

Remarks:

SAS Institute, Inc. 1999 - 2001. The SAS System for Windows. Version 8.2. Cary, North Carolina.

Value:

15.9

Remarks on result:

other: Non-edible tissue

Remarks:

5 ug/L nominal test article in water

Key result

Rate constant:

overall uptake rate constant (L kg-1 d-1)

Remarks:

SAS Institute, Inc. 1999 - 2001. The SAS System for Windows. Version 8.2. Cary, North Carolina.

Value:

9.1

Remarks on result:

other: Whole Fish w.w.

Remarks:

5 ug/L nominal test article in water

Key result

Rate constant:

overall uptake rate constant (L kg-1 d-1)

Remarks:

SAS Institute, Inc. 1999 - 2001. The SAS System for Windows. Version 8.2. Cary, North Carolina.

Value:

2.1

Remarks on result:

other: Edible tissue

Remarks:

50 ug/L nominal test article in water

Key result

Rate constant:

overall uptake rate constant (L kg-1 d-1)

Remarks:

SAS Institute, Inc. 1999 - 2001. The SAS System for Windows. Version 8.2. Cary, North Carolina.

Value:

16.9

Remarks on result:

other: Non-edible tissue

Remarks:

50 ug/L nominal test article in water

Key result

Rate constant:

overall uptake rate constant (L kg-1 d-1)

Remarks:

SAS Institute, Inc. 1999 - 2001. The SAS System for Windows. Version 8.2. Cary, North Carolina.

Value:

9.7

Remarks on result:

other: Whole fish w.w.

Remarks:

50 ug/L nominal test article in water

Key result

Rate constant:

overall depuration rate constant (d-1)

Remarks:

Calculated using SAS

Value:

0.046

Remarks on result:

other: Edible tissue

Remarks:

5 ug/L nominal test article in water

Key result

Rate constant:

overall depuration rate constant (d-1)

Remarks:

Calculated using SAS

Value:

0.044

Remarks on result:

other: Nonedible tissue

Remarks:

5 ug/L nominal test article in water

Key result

Rate constant:

overall depuration rate constant (d-1)

Remarks:

Calculated using SAS

Value:

0.045

Remarks on result:

other: Whole fish w.w.

Remarks:

5 ug/L nominal test article in water

Key result

Rate constant:

overall depuration rate constant (d-1)

Remarks:

Calculated using SAS

Value:

0.038

Remarks on result:

other: Edible tissue

Remarks:

50 ug/L nominal test article in water

Key result

Rate constant:

overall depuration rate constant (d-1)

Remarks:

Calculated using SAS

Value:

0.045

Remarks on result:

other: Nonedible tissue

Remarks:

50 ug/L nominal test article in water

Key result

Rate constant:

overall depuration rate constant (d-1)

Remarks:

Calculated using SAS

Value:

0.044

Remarks on result:

other: Whole fish w.w.

Remarks:

50 ug/L nominal test article in water

Details on kinetic parameters:

The kinetic uptake and depuration rate constants were determined using nonlinear regression described by Newman (6). In the sequential method, data from the depuration (elimination) phase was used to first estimate k2, and then using both the k2 estimate and fish tissue from the uptake phase to estimate k1. In addition, lipid-normalized and growth-corrected kinetic and steady state bioconcentration factors (BCFSSL, BCFKL, BCFKg and BCFKLG) as well as growth corrected half-life (t½g) were determined. All parameters calculated are presented in the attached table (Depuration Parameter Table) and the equations used are listed in Appendix 7.

Other kinetic parameters are listed in Tables 13, 14, 15 and 16.

Metabolites:

EXP1200078 is an UVCB material of higher molecular weight; the material is very viscous, tar-like and is not soluble in water or common non-aqueous solvents.
Preliminary efforts were made to detect individual components of the unlabeled and radiolabeled materials. Without standards of the individual UVCB
components, it was not possible to detect or characterize possible metabolites (even with a radiolabel).

Results with reference substance (positive control):

Since EXP1200078 is an UVCB substance, the complex chemistry precludes the abilitiy to synthesize reference substances. Therefore, there are no results with reference substances.

Details on results:

Other rate constants in tables 15 and 16.

Other figures, tables and appendices inserted as PDF in attached background material as needed.

Figure	Content
1	Concentrations of 14C-Test Substance in Edible Tissues of Bluegill in the Low Treatment Group
2	Concentrations of 14C-Test Substance in Non-edible Tissues of Bluegill in the Low Treatment Group
3	Concentrations of 14C-Test Substance in Whole Fish Tissues of Bluegill in the Low Treatment Grou
4	Concentrations of 14C-Test Substance in Edible Tissues of Bluegill in the High Treatment Group
5	Concentrations of14C-Test Substance in Non-edible Tissues of Bluegill in the High Treatment Group
6	Concentrations of 14C-Test Substance in Whole Fish Tissues of Bluegill in the High Treatment Group

Table	Content
00	Rate Constants Parameters Table
1	Measured Concentrations of the Test Substance in 14C Primary Stock Solutions
13	Steady-State BCF Values for Bluegill Based on Total Radioactivity
14	Kinetic Estimates of the BCFK for Bluegill Based on Total Radioactivity
15	Growth Rate Constants
16	Growth Corrected Values in Whole Fish Tissues
17	Whole Fish Lipid Determination

Appendix	Content
3	Analyses of Pesticides, Organics and Metals in Sera Vipan Feed Sample
4	Analyses of Pesticides, Organics and Metals in Wildlife International Well Water
5	Dilution Water Chemistry Parameters for the 4-Week Period Immediately Preceding the Study
6.2	Method Flowchart for the Analysis of 14C-Test Substance in Freshwater and Fish Tissue
6.4	Analytical Method Flowchart for Lipid Extraction from Fish Tissue
6.5	Edible and Non-edible Lipid Determination
6.6	HPLC Operational Parameters for Fractionation
7	Equations
16	Statistical Evaluation of Slopes for Growth Data
17	Results of a 96-Hour Static-Renewal Acute Toxicity Rangefinding Test with the Bluegill (Lepomis macrochirus)

Validity criteria fulfilled:

yes

Conclusions:

Values for all BCF parameters (steady-state and kinetic); edible, non-edible, whole-fish; and lipid-corrected parameters were below 500. Values were determined at two concentrations (4.8 ug/L and 48 ug/L). Because of the UVCB nature of the test material and very low aqueous solubility, it was not possible to develop analytical methods for the determination of individual components of the UVCB material.

Executive summary:

The results of the OECD 305 study conducted with the bluegill (Lepomis macrochirus) at two different concentrations demonstrate that the test substance designated as EXP1200078 does not bioaccumulate.

Description of key information

A key GLP study conducted according to OECD guideline determined that the registration substance is not bioaccumulative, with a BCF value of <500.

Key value for chemical safety assessment

BCF (aquatic species):

55 dimensionless

Additional information

The kinetic BCF for edible fish tissue at the highest exposure concentration of 48 ug/L. Even though the BCF for whole fish was the highest value (364) it was deemed that the value for edible fish (55) was the most relevant for human health assessment.