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Environmental fate & pathways

Bioaccumulation: aquatic / sediment

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Reference
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 21 to March 3, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD and US EPA test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1730 (Fish Bioconcentration Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
None specified
Radiolabelling:
no
Details on sampling:
Collection of Water Samples
Water samples were collected from the solvent control and each of the 3.5 and 35 μg/L treatment groups, 5 and 2 days prior to the start of the exposure period to confirm the concentrations after conditioning the diluter system. In the solvent control and both treatment groups, water samples were collected on days 0 (hours 0 and 4), 1, 3, 7, 14, and 21 of the uptake phase, and days 1, 3, 5, and 7 of the depuration phase for the analysis of Reofos 35. On each sampling day, one water sample was analyzed from the control test chamber and two from each treatment group test chambers. All water samples were collected in 20-mL preconditioned 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 23 of the uptake phase from the control and both treatment groups. Water samples were then stored frozen for possible analysis of metabolites.

Collection of Tissue Samples
Tissue samples were collected from the solvent control and both treatment groups on uptake days 0 (hour 4), 1, 3, 7, 14 and 21 and on depuration days 1, 3, 5 and 7. At each tissue sampling interval, a sufficient number of fish were collected to provide at least four replicate samples of solvent control fish and four replicate samples from each Reofos 35 treatment group. 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.

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, two fish were analyzed for lipid content from the control and each treatment group on day 0 of the uptake phase, one fish was analyzed for lipid content from the control and each treatment group on Day 23 of uptake and Day 10 of depuration 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 23 uptake) and at the end of depuration (Day 10 depuration). All fish collected for lipid content were stored frozen until analysis.
Vehicle:
yes
Details on preparation of test solutions, spiked fish food or sediment:
A primary stock solution of Reofos 35 was prepared by mixing a calculated amount (0.1610 g) of test substance into HPLC-grade DMF (460 mL) at a nominal concentration of 0.35 mg/mL. The primary stock was prepared four times throughout the study. The primary stock solutions were mixed by inversion and were clear and colorless with no visible precipitates. One secondary stock solution (410 mL) was prepared in DMF at a nominal concentration of 0.035 mg/mL by proportional dilution of the primary stock. The secondary stock solution was mixed by inversion and appeared clear and colorless with no visible precipitates.
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 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 3.5 and 35 μg/L. The solvent control was prepared by delivering DMF to the mixing chamber for the solvent control. The concentration of DMF in the solvent 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 9 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, with no evidence of precipitation observed in any control or treatment solution.
Test organisms (species):
Lepomis macrochirus
Details on test organisms:
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 May 12, 2014 (fish were approximately 8 months old at test initiation). 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 4.8 ± 0.40 cm (4.2 – 5.6 cm) and 1.42 ± 0.352 g (0.93 – 2.10 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 21.1 to 22.6°C, measured with a hand-held liquid-in-glass thermometer. The pH of the water ranged from 8.3 to 8.6, 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 one to three at a time to the test chambers until the solvent control and each treatment level test chamber contained 80 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 0.23 g fish/L/day. Instantaneous loading (the total wet weight of fish per liter of water in the tank) was 1.4 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 faeces 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.

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 um, and pumped into a 37,800-L storage tank and aerated with spray nozzles. Prior to use, the water was filtered to 0.45 um 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 of the report and are considered appropriate.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, and are considered appropriate.
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
23 d
Total depuration duration:
10 d
Hardness:
Hardness (mg/L as CaCO3)
141 (Mean) (N = 4)
136 – 144 (Range)
Test temperature:
Uptake: 21.4 – 21.8°C
Depuration: 21.4 – 22.0°C
pH:
8.0 (Mean) (N = 4)
8.0 – 8.0 (Range)
Dissolved oxygen:
Uptake Phase (mg/L) (A dissolved oxygen concentration of 5.2 mg/L represents 60% saturation in freshwater at 22°C.)
6.6 – 8.7 mg/L

Depuration Phase (mg/L) (A dissolved oxygen concentration of 5.2 mg/L represents 60% saturation in freshwater at 22°C.)
8.6 – 8.7 mg/L
TOC:
(mg C/L) <1 (N=1)
Salinity:
Not applicable - freshwater
Conductivity:
Not specified
Details on test conditions:
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 solvent control to test chambers. A syringe pump (Harvard Apparatus, Holliston, Massachusetts) was used to deliver volumes of test substance stock solutions and dimethylformamide (DMF) for the solvent control to mixing chambers indiscriminately assigned to each treatment and the control. The stock solutions or solvent 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 solvent 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 17.4 cm. On day 23 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 10 days of depuration. The depth of the test water in one representative test chamber during depuration was approximately 23.6 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, faecal matter, algae and bacterial growth. Test chambers were identified by the project number and test concentration.

Biological Observations
Assessments of survival and general observation (behavioral and physical) were made daily in order to assess the health of the fish. Any mortalities, external abnormalities or abnormal behaviour (relative to controls) were noted if observed.

Environmental Conditions
Fluorescent light bulbs that emit wavelengths similar to natural sunlight were used for illumination of the test chambers. A photoperiod of 16 hours of light and 8 hours of dark was controlled with an automatic timer. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity at the surface of the water at the start of the uptake phase was 751 lux and 589 lux at the start of the depuration phase. Light intensity was measured using a SPER Scientific Model 840006C light meter.
The target test temperature during the test was 22 ± 1°C. Temperature was measured in the test chamber at the beginning and end of the test and approximately weekly intervals during the test using a liquid-in-glass thermometer. Temperature was also monitored continuously in the solvent control test chamber using an Amegaview Central Monitoring system, which was verified prior to test initiation and at approximately weekly intervals thereafter using a digital thermometer and a liquid-in-glass thermometer, respectively.
Dissolved oxygen measurements were made in each test chamber daily during the uptake and depuration phases using a Thermo Orion Model 850Aplus dissolved oxygen meter. Measurements of pH were made in each test chamber at the beginning and end of the test and approximately weekly during the test using a Thermo Orion Model 525Aplus pH meter.
Hardness, alkalinity and conductivity were measured in the dilution water, the solvent control, and high treatment level at the beginning and end of the test, and approximately weekly during the test. Total organic carbon (TOC) was measured in the dilution water, the solvent control, low treatment level and high treatment level at the beginning and end of the test, and approximately weekly during the test. Hardness and alkalinity were measured by titration based on procedures in Standard Methods for the Examination of Water and Wastewater (4). Conductivity was measured using an Acorn Series CON 6 conductivity meter. Total organic carbon was measured using a Shimadzu model TOC-VCSH total organic carbon analyzer.
Nominal and measured concentrations:
3.5 μg/L; 35 μg/L (nominal). Measured concentrations are presented in table form - see Any other information
Reference substance (positive control):
no
Details on estimation of bioconcentration:
The steady-state bioconcentration factor (BCF) values were determined from the mean tissue concentrations at apparent steady-state divided by the average water concentration. Tissue concentrations were considered to be at apparent steady-state if three or more consecutive sets of tissue concentrations are within ±20% of each other, and there is no significantly increasing trend in residue concentration in time between the first and last successive analyses (p > 0.05). All statistical tests were performed using a personal computer with The SAS System for Windows software. Nonlinear regression was used to sequentially solve for k1 and k2 using fish tissue data from the uptake phase and depuration phase. These rate constants were used to calculate a kinetic bioconcentration factor (BCFK = k1/k2) and also to calculate half-life for clearance in tissue (t½) and, time to reach 95% of steady state (t95).
The kinetic uptake and depuration rate constants were determined using nonlinear regression described by Newman. 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.

Substance Uptake and Depuration Rate Constants and Bioconcentration Factors (BCF)
kg (growth rate constant; day-1)
k1 (overall uptake rate constant; L kg-1 day-1)
k2 (overall depuration rate constant; day-1)
k2g (growth-corrected depuration rate constant, day-1)
Cr (chemical concentration in the fish at steady-state; μg kg-1)
Cw (chemical concentration in the water; μg L-1)
Ln (lipid normalization factor)
BCFSS (steady-state BCF; L kg-1)
t1/2 (half-life, day)
t95 (time to reach 95% of steady state)
BCFSSL (lipid-normalized steady-state BCF; L kg-1)
BCFK (kinetic BCF; L kg-1)
BCFKg (growth-corrected kinetic BCF; L kg-1)
t1/2g (growth-corrected half-life; day)
BCFKL (lipid-normalized kinetic BCF: L kg-1)
BCFKLG (lipid-normalized growth-corrected kinetic BCF; L kg-1)
Lipid content:
>= 7.24 - <= 8.83 %
Time point:
end of exposure
Remarks on result:
other: Edible & non-edible fraction
Conc. / dose:
3.1 µg/L
Type:
BCF
Value:
225 dimensionless
Basis:
edible fraction
Time of plateau:
17.9 d
Calculation basis:
steady state
Remarks on result:
other: Conc.in environment / dose:3.1
Conc. / dose:
3.1 µg/L
Type:
BCF
Value:
281 dimensionless
Basis:
edible fraction
Time of plateau:
17.9 d
Calculation basis:
kinetic
Remarks on result:
other: Conc.in environment / dose:3.1
Conc. / dose:
3.1 µg/L
Type:
BCF
Value:
773 dimensionless
Basis:
non-edible fraction
Time of plateau:
7.2 d
Calculation basis:
steady state
Remarks on result:
other: Conc.in environment / dose:3.1
Conc. / dose:
3.1 µg/L
Type:
BCF
Value:
733 dimensionless
Basis:
non-edible fraction
Time of plateau:
7.2 d
Calculation basis:
kinetic
Remarks on result:
other: Conc.in environment / dose:3.1
Conc. / dose:
3.1 µg/L
Type:
BCF
Value:
512 dimensionless
Basis:
whole body w.w.
Time of plateau:
8.9 d
Calculation basis:
steady state
Remarks on result:
other: Conc.in environment / dose:3.1
Conc. / dose:
3.1 µg/L
Type:
BCF
Value:
516 dimensionless
Basis:
whole body w.w.
Time of plateau:
8.9 d
Calculation basis:
kinetic
Remarks on result:
other: Conc.in environment / dose:3.1
Conc. / dose:
24 µg/L
Type:
BCF
Value:
293 dimensionless
Basis:
edible fraction
Time of plateau:
4.9 d
Calculation basis:
steady state
Remarks on result:
other: Conc.in environment / dose:24
Conc. / dose:
24 µg/L
Type:
BCF
Value:
311 dimensionless
Basis:
edible fraction
Time of plateau:
4.9 d
Calculation basis:
kinetic
Remarks on result:
other: Conc.in environment / dose:24
Conc. / dose:
24 µg/L
Type:
BCF
Value:
922 dimensionless
Basis:
non-edible fraction
Time of plateau:
3.7 d
Calculation basis:
steady state
Remarks on result:
other: Conc.in environment / dose:24
Conc. / dose:
24 µg/L
Type:
BCF
Value:
776 dimensionless
Basis:
non-edible fraction
Time of plateau:
3.7 d
Calculation basis:
kinetic
Remarks on result:
other: Conc.in environment / dose:24
Elimination:
yes
Parameter:
DT50
Depuration time (DT):
4.13 d
Details on kinetic parameters:
Reported in table form - see Any other information
Results with reference substance (positive control):
Not applicable - reference substance not used in this study.
Details on results:
Concentrations of Reofos 35 in Water
The measured concentrations of Reofos 35 in the pretest samples collected 5 days prior to initiation were approximately 89 to 97% of nominal concentrations. Aeration was added to the test system 5 days prior to test initiation, after which the mean measured concentrations were 72 to 92% of nominal.
The measured concentrations ranged from 63.7 to 108% of the nominal concentration in the low level. Measured concentrations ranged from 48.9 to 85.7% of the nominal concentration in the high level. This resulted in an overall mean measured percent of nominal of 89% in the low level and 67% in the high level. The diluter system was checked one to two times daily and calibrated weekly during the study and indicated that the correct test concentrations were being delivered to the test chambers. The results of the study were based on the mean measured test concentrations for the uptake phase of 3.1 and 24 μg/L.
The measured concentrations in water during the depuration phase were below the LOQ in the low and high levels.

Concentrations of Reofos 35 in Fish Tissues
The mean measured tissue concentration of Reofos 35 of fish exposed to a mean measured test concentration of 3.1 μg/L edible, non-edible and whole fish at Day 21 of uptake was 701, 2408 and 1595 μg/kg, respectively.
The mean measured tissue concentration of Reofos 35 of fish exposed to a mean measured test concentration of 24 μg/L in edible, non-edible and whole fish at Day 21 of uptake was 6918, 21725 and 14953 μg/kg, respectively.

Determination of Steady State
It was determined the study had reached steady state on days 7, 14 and 21 in the low and high treatment groups. The steady-state BCF values for the low level (3.1 μg/L), based on Reofos 35 concentrations in tissue and water were 225, 773 and 512 in edible, non-edible and whole fish tissue, respectively. The steady-state BCF values for the high level (24 μg/L), based Reofos 35 concentrations in tissue and water were 293, 922 and 634 in edible, non-edible and whole fish tissue, respectively.

BCFK and Depuration Characteristics
Using nonlinear regression, similar kinetic BCF (BCFK) values were determined from total radioactivity data resulting in BCFK values of 281, 733 and 516 in edible, non-edible and whole fish tissue, respectively for the low level and 311, 776 and 559 in edible, non-edible and whole fish tissue, respectively for the high level. The estimated time to reach 95% of steady state (t95) in edible, non-edible and whole fish was 17.9, 7.2 and 8.9 days, respectively for the low level and 4.9, 3.7 and 4.0 days, respectively for the high level.
The mean measured concentration of total radioactivity in edible, non-edible and whole fish by Day 7 of depuration was
Growth Rate
Growth rate constants (kg), were calculated by performing a linear least squares correlation on the individual data of the two treatment groups. The slopes of the linear regression were compared statistically using the student’s t-test (p = 0.05).
There were no statistical differences between the slopes of the uptake and depuration phase growth data in the 3.1 μg/L treatment group (p > 0.05), therefore the uptake and depuration data was pooled to estimate the growth rate constant (kg). The growth rate constant (kg) for the 3.5 μg/L treatment group was -0.00797. In the 3.1 μg/L treatment group, the growth corrected depuration rate constant (k2g) was 0.3460, the growth corrected half-life (t1/2g) was 2.0 days and the growth corrected kinetic BCF (BCFKg) in whole fish tissue was 504.
There were no statistical differences between the slopes of the uptake and depuration phase growth data in the 24 μg/L treatment group (p > 0.05), therefore the uptake and depuration data was pooled to estimate the growth rate constant (kg). The uptake growth data was used to estimate the growth rate constant (kg). The growth rate constant (kg) for the 24 μg/L treatment group was -0.00681. In the 24 μg/L treatment group, the growth corrected depuration rate constant (k2g) was 0.7568, the growth corrected half-life (t1/2g) was 0.92 days and the growth corrected kinetic BCF (BCFKg) in whole fish tissue was 553.

Lipid Analysis
The mean measured percent lipid content in edible tissue from the fish sampled at the beginning of the test, Day 23 of uptake and Day 10 of depuration were 2.59, 2.26 and 2.80%, respectively. The mean measured percent lipid content in non-edible tissue from the fish sampled at the beginning of the test, Day 23 of uptake, Day 10 of depuration were 7.24, 8.83 and 8.57%, respectively. The lipid content in the edible and non-edible tissue remained fairly consistent over the course of the study.
The whole fish mean measured percent lipid content was calculated for each treatment group. Both the steady-state and kinetic BCF values for whole fish were normalized to 5% lipid content wet weight based on the procedures in the OECD 305 Guideline. Since there were no significant differences in growth between the treated and control fish, the lipid content of the control fish were used for lipid calculations. The mean lipid content of whole fish on Day 23 of the uptake phase from the solvent control (9.2%) was used in the lipid correction. The lipid-normalized steady state BCF (BCFSSL) in whole fish tissue was 278 and 344 in the low and high treatment groups, respectively. The lipid-normalized kinetic BCF (BCFKL) in whole fish tissue was 280 and 303 in the low and high treatment groups, respectively. The lipid normalized growth corrected kinetic BCF (BCFKgL) in whole fish tissue was 273 and 300 in the low and high treatment groups, respectively.

Physical and Chemical Measurements of Water
Water temperatures in the test chambers ranged from 21.4 to 22.0°C, and both manual and continuous temperature measurements were within the range of 22 ± 1°C established for the test. Measurements of water pH ranged from 7.9 to 8.4 during the test. Dissolved oxygen remained ≥ 6.6 mg/L (76% of saturation) throughout the test. Measurements of hardness, alkalinity and conductivity were typical of Wildlife International well water.
Total organic carbon (TOC) in the solvent control ranged from 5.304 to 54.12 mg C/L during uptake and was < 1 mg C/L during depuration. Total organic carbon (TOC) in the dilution water ranged from < 1 to 9.22 mg C/L during the uptake phase and ranged from < 1 to 1.607 mg C/L during the depuration phase. Total organic carbon (TOC) in the low treatment level ranged from 45.19 to 55.75 mg C/L during uptake and ranged from < 1 to 2.45 mg C/L during depuration. Total organic carbon (TOC) in the high treatment level ranged from 46.73 to 57.67 mg C/L during uptake and was < 1 mg C/L during depuration.

Observations of Mortality and Clinical Signs of Toxicity
There were no mortalties in the control or treatment groups during the study. At the end of the uptake phase (Day 23), the fish were enumerated during the transfer to depuration tanks.
All remaining fish appeared normal and healthy throughout the test.
Reported statistics:
None specified in the study report

Kinetic Estimates of the BCFK for Bluegill

Tissue Type

Nominal Exposure (μg/L)

Uptake Rate Constant (k1, Day-1)1

Depuration Rate Constant (k2, Day-1)1

Kinetic Bioconcentration Factor (BCFK) 2

Estimated Time to Reach 95% Steady State (Days)2

Estimated Time to Reach 50% Clearance (Days)2

Edible

3.5

47.1

0.168

281

17.9

4.13

Non-edible

3.5

303.9

0.414

733

7.2

1.67

Whole fish

3.5

174.4

0.338

516

8.9

2.05

Edible

35

188.4

0.607

311

4.9

1.14

Non-edible

35

629.2

0.811

776

3.7

0.86

Whole fish

35

418.8

0.750

559

4.0

0.92

1Calculated using SAS

2Calculated using methods outline in OPPTS guideline 850.1730

Mean Measured Concentrations of Reofos 35 in Edible, Non-edible and Whole Fish Tissues in the Low and High Exposure Groups by Day During the Uptake and Depuration Phases

Nominal Concentration Reofos 35 (μg/L)

Study Phase

Sampling Interval (Day)

Mean Edible Tissue Conc. (μg/kg)

Mean Non-Edible Tissue Conc. (μg/kg)

Mean Whole Fish Conc.1,2,3,4(μg/kg)

3.5

Uptake

0.17

<LOQ

<LOQ

<LOQ

3.5

Uptake

1

404

1066

750

3.5

Uptake

3

608

1605

1179

3.5

Uptake

7

711

1923

1345

3.5

Uptake

14

702

2297

1499

3.5

Uptake

21

701

2408

1595

35

Uptake

0.17

1340

3315

2396

35

Uptake

1

3195

9875

6702

35

Uptake

3

5933

13550

10189

35

Uptake

7

7848

19625

14112

35

Uptake

14

7273

16375

11838

35

Uptake

21

6918

21725

14953

3.5

Depuration

0

701

2408

1595

3.5

Depuration

1

<LOQ

845

485

3.5

Depuration

3

<LOQ

<LOQ

<LOQ

3.5

Depuration

5

<LOQ

<LOQ

<LOQ

3.5

Depuration

7

<LOQ

<LOQ

<LOQ

35

Depuration

0

6918

21725

14953

35

Depuration

1

2863

16050

9901

35

Depuration

3

509

4223

2573

35

Depuration

5

<LOQ

295

<LOQ

35

Depuration

7

<LOQ

<LOQ

<LOQ

1Whole Fish Conc. = [(edible wt. * edible conc.) + (nonedible wt. * nonedible conc.)] / (edible wt. + nonedible wt.)

2Results were generated using Excel 2010 and SAS. Manual calculations may differ slightly.

3LOQ = 250 μg/kg.

4When equivalents were <LOQ, ½ LOQ (125 μg/kg) was used for calculations

 

Confirmation of Steady State

Mean Measured Tissue Concentration (μg/kg)

Tissue Type

Nominal Water Concentration (μg/L)

For the Last Three Sampling Intervals

Mean

SD

p1

Day 7

Day 14

Day 21

Edible

3.5

711.0

702.0

701.0

704.7

5.51

0.2791

Non-Edible

3.5

1922.5

2296.7

2407.5

2208.9

254.14

0.0562

Whole Fish

3.5

1344.8

1498.5

1594.8

1479.4

126.08

0.1587

Edible

35

7847.5

7272.5

6917.5

7345.8

469.32

0.8792

Non-Edible

35

19625.0

16375.0

21725.0

19241.7

2695.52

0.2321

Whole Fish

35

14111.6

11838.5

14952.7

13634.2

1611.05

0.2791

1p = 0.05

 

Steady-State BCF Values for Bluegill

Tissue Type

Mean Measured Test Concentration (μg/L)

Uptake Days at Steady State

Mean Measured Steady-State Tissue Concentration (μg/kg)

Steady-State BCF

Edible

3.1

7-14-21

701.0

225

Non-Edible

3.1

7-14-21

2407.5

773

Whole Fish

3.1

7-14-21

1594.9

512

Edible

24

7-14-21

6917.5

293

Non-Edible

24

7-14-21

21725.0

922

Whole Fish

24

7-14-21

14952.7

634

 

Kinetic Estimates of the BCFK for Bluegill

Tissue Type

Nominal Exposure (μg/L)

Uptake Rate Constant (k1, Day-1) 1

Depuration Rate Constant (k2, Day-1)1

Kinetic Bioconcentration Factor (BCFK)2

Estimated Time to Reach 95% Steady State (Days)2

Estimated Time to Reach 50% Clearance (Days)2

Edible

3.5

47.1

0.168

281

17.9

4.13

Non-Edible

3.5

303.9

0.414

733

7.2

1.67

Whole Fish

3.5

174.4

0.338

516

8.9

2.05

Edible

35

188.4

0.607

311

4.9

1.14

Non-Edible

35

629.2

0.811

776

3.7

0.86

Whole Fish

35

418.8

0.750

559

4.0

0.92

1Calculated using SAS

2Calculated using methods outlined in OPPTS guideline 850.1730

 

Growth Rate Constants

Kg values

 

3.1 μg/L1,2

24 μg/L1,2

Uptake

-0.00569

-0.00339

Depuration

0.00192

-0.00330

Pooled

-0.00797

-0.00681

1No statistical difference between slopes of uptake and depuration phases (p>0.05)

2Growth measurements from both the uptake and depuration phases were pooled for the overall growth rate constant.

Validity criteria fulfilled:
yes
Conclusions:
The substance is not considered to be bioaccumulative.
Executive summary:

The study was conducted according to the procedures outlined in the protocol, “Reofos 35: An Aqueous Exposure Bioaccumulation Test with the Bluegill (Lepomis macrochirus)”. The protocol was based on procedures outlined in U.S. Environmental Protection Agency Series 850 – Ecological Effects Test Guidelines, OPPTS Number 850.1730: Fish BCF; ASTM Standard E1022-94, Standard Guide for Conducting Bioconcentration Tests with Fishes and Saltwater Bivalve Molluscs and OECD Guideline for Testing of Chemicals 305, Bioaccumulation in Fish: Aqueous and Dietary Exposure.

 

The test was divided into two phases: uptake and depuration. During the uptake phase, bluegill were exposed to two test concentrations of Reofos 35 and a solvent control (dilution water containing 0.1 mL/L DMF). The nominal concentrations of 3.5 and 35 μg/L were selected in consultation with the Sponsor and were based on existing toxicity data and preliminary trials. Each group consisted of one test chamber with 80 fish in the solvent control and each treatment level test chamber. During the depuration phase, fish were exposed to dilution water only. The duration of the uptake phase was 23 days for both of the treatment groups. After confirmation of steady state, fish were moved into the depuration phase which lasted for 10 days for both treatment levels. During both phases of the test, test organisms and water samples were collected and analyzed for Reofos 35.

These values were used to determine the substance-specific half-life (t1/2), the kinetic bioconcentration factor (BCFK) and the steady state bioconcentration factor (BCFSS) for the test substance in fish, as well as lipid-normalized and growth-corrected kinetic and steady state bioconcentration factors (BCFSSL, BCFKL, BCFKgand BCFKLg).

 

Bluegill were exposed to mean measured water concentrations of 3.1 and 24 μg/L.

Steady-state concentrations of Reofos 35 were achieved in the tissues of Bluegill (Lepomis macrochirus) after 21 days of uptake in the low and high treatment groups. Steady-state BCF values for the low treatment group were 225, 773 and 512 in edible, non-edible and whole fish, respectively.

Steady-state BCF values for the high treatment group were 293, 922 and 634 in edible, non-edible and whole fish tissue, respectively. Reofos 35 quickly depurated from all fish tissue and by Day 7 of depuration tissue concentrations were approximately <LOQ in both treatment groups.

Kinetic BCFK values derived by nonlinear regression for the low treatment group were 281, 733 and 516 for edible, non-edible and whole fish tissue, respectively. The time to reach 95% steady state based on kinetics was 17.9, 7.2 and 8.9 days and time to reach 50% clearance was 4.1, 1.7 and 2.1 days for edible, non-edible and whole fish, respectively.

Kinetic BCFK values derived by nonlinear regression for the high treatment group were 311, 776 and 559 for edible, non-edible and whole fish tissue, respectively. The time to reach 95% steady state based on kinetics was 4.9, 3.7 and 4.0 days and time to reach 50% clearance was 1.1, 0.86 and 0.92 days for edible, non-edible and whole fish, respectively.

The substance is not considered to be bioaccumulative.

Description of key information

Discussions on bioaccumulation potential

Key value for chemical safety assessment

BCF (aquatic species):
776 L/kg ww

Additional information

An assessment of the propensity towards bioaccumulation was undertaken, utilizing an appropriate study as well as QSAR derivation, using recognized tools. The results are as follows:

 

Study data:

 

Bluegill were exposed to mean measured water concentrations of 3.1 and 24 μg/L. Steady-state concentrations of Reofos 35 were achieved in the tissues of Bluegill (Lepomis macrochirus) after 21 days of uptake in the low and high treatment groups. Steady-state BCF values for the low treatment group were 225, 773 and 512 in edible, non-edible and whole fish, respectively. Steady-state BCF values for the high treatment group were 293, 922 and 634 in edible, non-edible and whole fish tissue, respectively. Reofos 35 quickly depurated from all fish tissue and by Day 7 of depuration tissue concentrations were approximately <LOQ in both treatment groups.

Kinetic BCFK values derived by nonlinear regression for the low treatment group were 281, 733 and 516 for edible, non-edible and whole fish tissue, respectively. The time to reach 95% steady state based on kinetics was 17.9, 7.2 and 8.9 days and time to reach 50% clearance was 4.1, 1.7 and 2.1 days for edible, non-edible and whole fish, respectively.

 

Kinetic BCFK values derived by nonlinear regression for the high treatment group were 311, 776 and 559 for edible, non-edible and whole fish tissue, respectively. The time to reach 95% steady state based on kinetics was 4.9, 3.7 and 4.0 days and time to reach 50% clearance was 1.1, 0.86 and 0.92 days for edible, non-edible and whole fish, respectively.

 

The substance is not considered to be bioaccumulative.

 

Kinetic Estimates of the BCFK for Bluegill

 

Tissue Type

Nominal Exposure (μg/L)

Uptake Rate Constant (k1, Day-1)1

Depuration Rate Constant (k2, Day-1)1

Kinetic Bioconcentration Factor (BCFK) 2

Estimated Time to Reach 95% Steady State (Days)2

Edible

3.5

47.1

0.168

281

17.9

Non-edible

3.5

303.9

0.414

733

7.2

Whole fish

3.5

174.4

0.338

516

8.9

Edible

35

188.4

0.607

311

4.9

Non-edible

35

629.2

0.811

776

3.7

Whole fish

35

418.8

0.750

559

4.0

 

QSAR derivation, using recognized tools

·       BCF model (CAESAR) (version 2.1.11)

·       BCF model (Meylan) (version 1.0.0)

·       BCF Read-Across (version 1.0.0)

·       US EPA On-Line EPI Suite™v4.0 model BCFBAF

 

It is understood that Annex  XI, section  1.3  allows  adaptation of the standard testing requirements by making use of (Q)SAR only if the following conditions are met:

 

(i) results are derived from   a  (Q)SAR  model  whose  scientific validity has  been  established,

(ii) the substance falls within the applicability domain of the (Q)SAR model,

(iii) results are adequate for the purpose of classification and labelling and/or risk assessment, and

(iv) adequate and  reliable documentation of  the  applied method is  provided.

 

 Within the four models used, the conditions (i) through (iv) are considered to be met. As the substance has some 4096 potential structures, an assessment of 50 of these, taken at random, was undertaken. The structures assessed were deemed to mainly fall within the applicability domain of the model, and this is demonstrated within the relevant QPRF’s. The models are recognized, and are referenced within ECHA’s own guidance and/or have relevant QMRF’s which are also detailed.

 

As a result, the QSAR’s conducted all generally agree with each other that the substance is not bioaccumulative.

 

Whilst this mean result is presented as only a general indication, this supports the overall conclusion that the substance is not considered to be bioaccumulative. The substance is not considered to be “bioaccumulative” or “very bioaccumulative” on the basis of the available data.