Reaction products of bis(4-methylpentan-2-yl)dithiophosphoric acid with phosphorus oxide, propylene oxide and amines, C12-14-alkyl (branched)

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 April 2009 - 9 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to standard procedure study; well documented study report

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1730 (Fish Bioconcentration Test)

Deviations:

yes

Remarks:

1) a pretest estimate of k2 was not calculated; 2) the time to steady state was not estimated prior to test initiation; 3) the loading rate for the test was 1.10 g fish/L/day. The protocol specified a loading rate of 1.0 g fish/L/day.

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
The BCF study was conducted on a surrogate molecule of the UVCB substance. Because the chemical identity of this substance is very complex it would not lend itself to conventional tests for bioaccumulation potential. To avoid this situation, a stable molecule with strong similarity to several representative structures of the UVCB substance was synthesized so that reliable bioaccumulation testing could be performed. The surrogate structure is also the predominant constituent of the UVCB product.

-Name of test material (as cited in study report): Radiolabeled 14C-test material and non-radiolabeled test material

-Smiles structure: CC(C)CC(C)OP(SCC(O)C)(OC(C)CC(C)C)=S.C

- Physical state: liquid

- Storage condition of test material: test material was stored under ambient conditions; 14C-test material was stored under frozen conditions

- Other: 14C-test material had radiochemical purity of 99.3% and specific radioactivity of 59 mCi/mmol (2.18 GBq/mmol)

Radiolabelling:

yes

Details on sampling:

- Sampling intervals/frequency for test organisms:

Collection of Tissue Samples:
Tissue samples were collected during the uptake phase on days 0, 1, 3, 7, 14, 21, 24, 28 and 31 of the 35-day uptake period, and during the depuration phase on Days 0, 1, 3, 7, 10, 14, 23, 37 and 62 of the 62-day depuration period. At each tissue sampling interval, a sufficient number of fish were collected to provide at least two replicate samples of solvent control fish and four replicate samples of each OS242157 treatment group. Fish were impartially removed from the test chambers and euthanized by severing the spinal cord at the point just posterior to the base of the pectoral fin dorsally. The fish were blotted dry and measured for total length and wet weight within approximately 15 minutes of collection. Each fish was then dissected and divided into edible 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. Tissue samples were transferred to tared glass jars and weighed. All tissue
samples were processed immediately for analysis or stored frozen. Selected fish were collected to determine lipid content. Fish for lipid analysis were sampled prior to test initiation, on Day 35 of uptake phase and on Day 62 of depuration phase. All fish collected for lipid content were stored frozen until analysis. Since there was insufficient radioactivity in the samples for fractionation, no samples were collected and processed for the determination of parent material using fractionation by HPLC.



- Sampling intervals/frequency for test medium samples:

Collection of Water Samples:
Water samples were collected from each treatment and control group prior to the start of the test on days –12, -11, -8, -7 and –1. Water samples were also collected from each treatment and control group on Days 0, 1, 3, 7, 9, 14, 21, 24, 28, 31 and 35 of the uptake phase and on Days 1, 3, 7, 10, 14, 23, 37 and 62 of the depuration phase. All samples were collected from mid-depth, using glass volumetric pipette, placed in glass vials, and processed immediately for analysis of total radioactivity. Additional water samples were collected from each treatment and control group during pretest on Day –7 and on Days 0 and 9 of the uptake phase, and processed for the analysis of non-radiolabeled OS242157. The low recoveries results observed in the samples collected from both prior to and after the fish were introduced into the system indicated that the loss of material was likely due to adsorption of material to the test system. Therefore, no additional samples were collected for the analysis of non-radiolabeled OS242157
during the remaining of the test. Since there was insufficient radioactivity in the samples for fractionation, no samples were collected and processed for the determination of parent material using fractionation by HPLC. All samples were also collected from mid-depth, using glass volumetric pipette,
placed in glass vials, and processed immediately for analysis. At each sampling interval, one water sample was collected from the control group, while two water samples were collected from each of the treatment group, for a total of five water samples at each sampling interval. Additionally, on Day 35 of the uptake phase, two 1-L water samples were collected from each test chamber by siphoning from mid-depth into a graduated cylinder, transferred into a 2-L Nalgene® bottle, and stored frozen for possible future metabolite identification.

- Sample storage conditions before analysis: no data

Vehicle:

yes

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

-Preparation of Test Solutions:
One stock was prepared using the 14C labeled test material during the study by dissolving the test material in dimethylformamide (DMF). The radioactivity of the stock prepared was confirmed prior to use in the study by analyzing triplicate 20 μL samples of stock solution by liquid scintillation counting (LSC) . The stock was found to have a concentration of 121 mg/L. Five primary stock solutions of non-radiolabeled test material were prepared during the study on the same day dispensing stocks were prepared. The 1.0 mg/mL primary stock solutions were prepared in DMF by weighing a target amount of 0.050 g of test substance and bringing to a 50-mL final volume. The stocks were mixed by inversion or stirring. After mixing, the stock solutions appeared clear and colorless. Dispensing stock solutions were prepared at the concentrations of 0.007 and 0.07 mg/mL to achieve the target concentration of 0.25 and 2.5 mg/mL. An appropriate volume of the radiolabeled primary stock solution was added to each dispensing stock solution such that the radioactivity in the dispensing stock was approximately 350,000 dpm/mL in order to achieve a final nominal radioactivity in each of the aqueous exposure solutions of 35 dpm/mL. The remaining test material needed for each dispensing stock solution was added as the non-radiolabeled primary stock solution. The solvent DMF was added to bring each dispensing stock solution to a final volume to achieve the nominal dispensing stock solution concentration. The dispensing stock solutions were injected into the diluter mixing chambers (at a rate of 35 μL/minute) where they were mixed with well water (at a rate of 350 mL/minute) to achieve the desired aqueous test concentrations. The solvent control was achieved by injecting DMF into the diluter mixing chamber at the same rate. The concentration of DMF in the solvent control and all treatment groups during the uptake phase was 0.1 mL/L.

Test organisms (species):

other: Onchorhynchus mykiss

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Strain: Oncorhynchus mykiss
- Source: obtained from Thomas Fish Company of Anderson California and were hatched on April 27, 2009.
- Age at study initiation (mean and range, SD): All fish used in the test were from the same source and year class

- Feeding during test
- Food type: commercially prepared diet (Salmon Starter) supplied by Zeigler Brothers Inc (Gardners PA)
- Frequency: fish were not fed on the last day of the study

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): Same.

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

97 d

Total depuration duration:

62 d

Hardness:

The mean hardness for the 4 week period immediately preceding the study was 138 (N=4) with a range from 136 to 140 mg/L as CaCO3. Hardness in the solvent control during the testing period ranged from 102 to 142 mg/L CaCO3

Test temperature:

Temperature values ranged from 11.2 to 12.7 degrees C during the testing period.

pH:

The pH ranged from 8.1 to 8.2 in the four week period immediately preceding the study.

Dissolved oxygen:

Dissolved oxygen values ranged from 6.6 to 9.2 mg/L

TOC:

Mean TOC values in the solvent control were 44.5 mg C/L in the updaka phase and 1.12 in the depuration phase. Mean TOC values in the dilution water were 0.65 in the uptake phase and 0.66 in the depuration phase.

Salinity:

n/a freshwater used

Details on test conditions:

TEST SYSTEM
- Test vessel: Test chambers were 127 L Teflon lined stainless steel aquaria filled with 80 L of test solution. The depth of the test water during uptake was approximately 19.8 cm.
- Material, size, headspace, fill volume: 127 L Teflon lined stainless steel aquaria filled with 80L of test solution.
- Aeration: Continuous
- Type of flow-through (e.g. peristaltic or proportional diluter): Continuous flow-through diluter system. A syringe pump was used to deliver the test substance. The flow of dilution water was controlled by rotameters.
- Renewal rate of test solution (frequency/flow rate): Approximately 6 volume additions of test water were delivered to the test chambers every 24 hours.
- No. of organisms per vessel:
90 fish per test chamber
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1
- Biomass loading rate: 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 1.1 g fish/L/day. Instantaneous loading (the total wet weight of fish per liter of water in the tank) was 6.92 g/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for holding and testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International, Ltd. 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 again was filtered (0.45 μm) to remove microorganisms and particles. Periodic analyses was performed to measure the concentrations of selected contaminants in well water. Hardness, alkalinity, pH and specific conductance were measured weekly to monitor the consistency of the well water.

OTHER TEST CONDITIONS
- Adjustment of pH: Measurements of pH were made in each test chamber at the beginning and end of the test and approximately weekly during the test.
- Photoperiod: 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: Fluorescent light bulbs that emit wavelengths similar to natural sunlight were used for
illumination of the test chambers. Light intensity at the surface of the water at the start of the
uptake phase was 342 lux and was 303 lux at the start of the depuration phase. Light intensity was measured using a SPER Scientific Model 840006C light meter.


RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: A 96 hour flow through acute toxicity testing with rainbow trout following OECD 203 and EPA 850.1075 guidelines was conducted with the rainbow trout. Test concentrations included a negative control, solvent control, and 5 nominal and measured concentrations of the test substance ranging from 88 ug/L to 1400 ug/L. Based on the mean measured test concentrations the 96 hour LC50 was 732 ug/L (550-973 95% CI) and the NOEC was 305 ug/L.

Nominal and measured concentrations:

Target concentrations were 0.25 ug/L and 2.5 ug/L; Mean measured concentrations were 0.25 ug/L and 2.8 ug/L.

Reference substance (positive control):

no

Details on estimation of bioconcentration:

The BCFK can be defined as the ratio of the uptake rate constant, k1 to the elimination (depuration) rate constant, k2:

BCFK = k1/k2

This kinetic definition of the BCFK is valid regardless of whether a steady-state concentration of a chemical is achieved during uptake as long as first-order kinetics can be assumed for uptake and elimination processes. Both the uptake rate, k1, and elimination rate, k2, of the chemical can be estimated by measuring concentrations of the chemical in water and fish tissues at several points in time. If elimination follows first order kinetics, the decline in log concentration of the chemical in tissues over time is a straight line. The slope of this line is the elimination rate constant, k2.
Thus, if measurements of chemical concentration in tissues are made at only two time points, the estimate can be obtained using the simple algebraic relationship:

k2 = (lnCm –lnCt) / te


Alternatively, k2. can be estimated as the slope calculated by linear regression if measurements are made at more than two time points. Finally, the uptake rate is calculated as where t is the length of the uptake (exposure) period and Cw is the concentration of OS242157 in the water during the exposure period:

k1 = k2Cm/Cw(1-e^-k2t)

Lipid content:

3.76 %

Time point:

other: during pretest

Remarks on result:

other: lipid content in edible tissue from fish sampled during pretest

Lipid content:

3.78 %

Time point:

other: day 35 of upate

Remarks on result:

other: lipid content in edible tissue from fish

Lipid content:

2.87 %

Time point:

other: day 62 of depuration

Remarks on result:

other: lipid content in edible tissue from fish

Lipid content:

8.8 %

Time point:

other: during pretest

Remarks on result:

other: lipid content in non-edible tissue from fish

Lipid content:

11.3 %

Time point:

other: day 35 of uptake

Remarks on result:

other: lipid content in non-edible tissue from fish

Lipid content:

10.1 %

Time point:

other: day 62 of depuration

Remarks on result:

other: lipid content in non-edible tissue from fish

Type:

BCF

Value:

436 L/kg

Basis:

whole body w.w.

Time of plateau:

35 d

Calculation basis:

steady state

Remarks on result:

other: Conc.in environment / dose:0.25 ug/L mean measured concentration in water

Type:

BCF

Value:

432 L/kg

Basis:

whole body w.w.

Time of plateau:

35 d

Calculation basis:

steady state

Remarks on result:

other: Conc.in environment / dose:2.8 ug/L mean measured concentration in water

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

23.83 d

Elimination:

yes

Parameter:

DT90

Depuration time (DT):

79.1 d

Details on kinetic parameters:

Kinetic parameters (details in Table 2)

- Uptake rate constant (k1) at nominal exposure of 0.25 ug/L for whole fish was 24.3

-Uptake rate constant (k1) at nominal exposure of 2.5 ug/L for whole fish was 29.6

- Depuration (loss) rate constant (k2) at nominal exposure of 0.25 ug/L for whole fish was 0.029

- Depuration (loss) rate constant (k2) at nominal exposure of 2.5 ug/L for whole fish was 0.030

- Computation / data analysis: Uptake rate constant (k1) caluclated using SAS. Depuration rat constant (k2) calculated using methods outlined in OPPTS guideline 850.1730

Metabolites:

No data. On Day 35 of the uptake phase, two 1-L water samples were collected from each test chamber by siphoning from mid-depth into a graduated cylinder, transferred into a 2-L Nalgene bottle, and stored frozen for possible future metabolite identification

Results with reference substance (positive control):

Not applicable

Details on results:

The measured concentrations of test material in the pretest samples collected 12 and 11 days prior to initiation were 66 and 69% of the nominal concentrations, respectively.The measured concentrations of 14C-test material ranged from approximately 78 to 112% of the target concentration in the low treatment group and 77 to 136% of the target concentration in the high treatment group. This resulted in an overall mean measured percent of target of 100 and 112% in the low and high treatment groups, respectively.

Water samples were collected to determine the concentrations of non-radiolabeled test material in water 7 days prior to test initiation to verify the operation of the diluter. However, the recoveries of the analytical results were low, which indicated a possible loss of material to adsorption to the test system, and the results were not reported. Measured concentrations of non-radiolabeled test material in water samples collected during the uptake phase on Days 0 and 9. The measured concentrations of non-radiolabeled test material ranged from approximately 39.4 to 53.3% of the target concentration in the 0.25 μg/L treatment group, and 61.7 to 67.2% of the target concentration in the 2.5 μg/L treatment group. The low recoveries results were likely due to the affinity of the test material to the test system. Since the analytical method for non-radiolabeled test material in fish tissues was not available at the time, the analytical results of the non-radiolabeled in water alone added no additional information to the study. Therefore, the decision was made to discontinue the analysis of nonradiolabeled test material in the water during the remaining of the test.

The mean measured tissue concentration of 14C-test material in edible, non-edible and whole fish at Day 31 of uptake was 77, 159 and 111 μg/kg, respectively. The steady-state BCF value based on total radioactivity and expressed as μg test material equivalents/L was 301, 625 and 436 in edible, non-edible and whole fish tissue, respectively. The mean measured concentration of 14C-test material in edible, non-edible and whole fish by Day 62 of depuration was 14, 16 and 15 μg/kg, respectively, which is approximately 18, 10 and 13% of the mean tissue concentration at Day 31 of uptake.

The mean measured tissue concentration of 14C-test material in edible, non-edible and whole fish at Day 31 of uptake was 719, 1859 and 1194 μg/kg, respectively. The steady-state BCF value based on total radioactivity and expressed as μg test material equivalents/L was 260, 673 and 432 in edible, non-edible and whole fish tissue, respectively. The mean measured concentration of 14C-test material in edible, non-edible and whole fish by Day 62 of depuration was 134, 215 and 162 μg/kg, respectively, which is approximately 19, 12 and 14% of the mean tissue concentration at Day 31 of uptake.

The mean measured percent lipid content in edible tissue from the fish sampled during pretest, Day 35 of uptake, and Day 62 of depuration was 3.76, 3.78 and 2.87%, respectively. The mean measured percent lipid content in non-edible tissue from the fish sampled during pre-test, Day 35 of uptake and Day 62 of depuration was 8.80, 11.3 and 10.1%, respectively. The lipid content in both the edible and non-edible tissue remained approximately the same over the course of the study. All test solutions appeared clear and colorless in the mixing chambers and test chambers at test initiation and termination during both the uptake and depuration phases.

There were no mortalities in any of the treatment groups; however one fish from the solvent control and
one fish from the 2.5 μg/L treatment groups were removed from the test after being exposed to the water
bath and no longer affording reliable study data. All remaining fish appeared normal and healthy
throughout the test.

The test met with the following criteria used to judge the validity of the test:

1. the temperature variation was less than ± 2°C during the test;

2. dissolved oxygen concentration in test solution remained >60% of air saturation during the test; and

3. the mortality and/or sublethal effects in the control and treatment groups were <10% at the
end of the test.

Reported statistics:

no data

Results shown in attached figures.

Validity criteria fulfilled:

yes

Conclusions:

Steady-state concentrations of 14C-test material were achieved in the tissues of rainbow trout (Oncorhynchus mykiss) after 31 days. The mean measured water concentrations based on total radioactivity were 0.25 and 2.8 μg/L. Steady-state BCF values for the 0.25 μg/L test concentration, based
on total radioactivity test material concentrations were 301, 625 and 436 in edible, non-edible and whole fish tissue, respectively. Steady-state BCF values for the 2.8 μg/L test concentration, based on total radioactivity test material concentrations were 260, 673 and 432 in edible, non-edible and whole fish tissue, respectively. Test material depurated slowly from fish tissue and consequently the study was terminated after reaching 62 days depuration, even though concentrations in the fish tissues remained above 10% of the steady state values.

Because the declining concentrations in the tissues did not follow the first order assumptions kinetics required for valid kinetic estimates of BCF, the results of the derived kinetic BCF values were questionable and the steady state estimates of the BCF was considered to be the most accurate of the available BCF estimates from this study.

Executive summary:

Methods.      

The bioconcentration study in rainbow trout was conducted according US EPA OPPTS 850.1730 and OECD 305 Guidelines.

 

Supply of test water: Continuous flow-through diluter system The length of exposure was 97 days with 35 days uptake and 62 days depuration.

The target concentrations were solvent control, 0.25 ug/L and 2.5 ug/L. The mean measured concentrations were <LOQ, 0.25 ug/L and 2.8 ug/L for the solvent control, low exposure level, and high exposure level respectively.

 

Results

Based on the mean measured concentrations in water the Steady state BCF for the whole fish at the low exposure level was 426 (266-619 95% CI) and the steady state BCF for the whole fish in the high exposure level was 432 (210-571 95% CI).

Discussion

Steady-state concentrations of 14C-test material were achieved in the tissues of rainbow trout (Oncorhynchus mykiss) after 31 days. The mean measured water concentrations based on total radioactivity were 0.25 and 2.8 μg/L. Steady-state BCF values for the 0.25 μg/L test concentration, based on total radioactivity test material concentrations were 301, 625 and 436 in edible, non-edible and whole fish tissue, respectively. Steady-state BCF values for the 2.8 μg/L test concentration, based on total radioactivity test material concentrations were 260, 673 and 432 in edible, non-edible and whole fish tissue, respectively. Test material depurated slowly from fish tissue and consequently the study was terminated after reaching 62 days depuration, even though concentrations in the fish tissues remained above 10% of the steady state values. Because the declining concentrations in the tissues did not follow the first order assumptions kinetics required for valid kinetic estimates of BCF, the results of the derived kinetic BCF values were questionable and the steady state estimates of the BCF was considered to be the most accurate of the available BCF estimates from this study.

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to standard procedure study; well documented study report

Reason / purpose for cross-reference:

read-across source

Type:

BCF

Value:

436 L/kg

Basis:

whole body w.w.

Time of plateau:

35 d

Calculation basis:

steady state

Remarks on result:

other: Conc in environment

Remarks:

dose 0.25 µ/L

Type:

BCF

Value:

432 L/kg

Basis:

whole body w.w.

Time of plateau:

35 d

Calculation basis:

steady state

Remarks on result:

other: Conc in environment

Remarks:

dose 2.8 µ/L mean

Description of key information

Steady state estimates of the BCF was considered to be the most accurate of the available BCF estimates from investigation of an analogue substance (US EPA OPPTS 850.1730 and OECD 305).

Key value for chemical safety assessment

BCF (aquatic species):

432 dimensionless

Additional information

A bioconcentration study in rainbow trout was conducted according US EPA OPPTS 850.1730 and OECD 305 Guidelines using an analogue substance. Supply of test water: Continuous flow-through diluter system The length of exposure was 97 days with 35 days uptake and 62 days depuration. The target concentrations were solvent control, 0.25 ug/L and 2.5 ug/L. The mean measured concentrations were <LOQ, 0.25 ug/L and 2.8 ug/L for the solvent control, low exposure level, and high exposure level respectively.

Based on the mean measured concentrations in water the Steady state BCF for the whole fish at the low exposure level was 426 (266-619 95% CI) and the steady state BCF for the whole fish in the high exposure level was 432 (210-571 95% CI).