bis(branched-alkyl) 2-{[(1-phenylethyl)amino]methyl}alkanedioate

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 May 2017 to 30 November 2017

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

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Details on sampling:

ANALYTICAL SAMPLING
- Homogeneity of the test substance in the treatment diet was evaluated by collecting nine samples from the test item fortified diet and control diet prior to initiation of the study.
- Samples of the treatment and control diets were also collected and analyzed in triplicate for the 14C test substance at the beginning and end of the uptake phase.
- The total lipid content of the prepared diet was sampled prior to the start of the test and at the end of the uptake phase.

TISSUE SAMPLING
- Tissue samples were collected on uptake day 14 and on depuration days 1, 2, 3, 4, 5, 6, and 11. At each tissue sampling interval, a sufficient number of fish were collected to provide at least six replicate samples of negative control fish and six replicate samples from the 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. The whole fish was transferred to pre-weighed glass vials and weighed. All tissue samples were processed immediately or stored frozen.
- On Days 0 and 14 of uptake and at the end of depuration (Day 12 depuration), additional fish were collected to determine lipid content. All fish collected for lipid content were stored frozen until analysis. 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 group. After sampling, the gut tract was removed from each fish. The separated gut tract and corresponding somatic (non-gut) tissue samples were transferred to pre-weighed glass vials. All tissues were processed immediately or stored frozen.

Vehicle:

no

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

DILUTION WATER
- The water used for organism holding and testing was freshwater obtained from a well approximately 40 meters deep located on the EAG Laboratories site in Easton, Maryland. 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 where the water was aerated with spray nozzles. Prior to use in the test system, the water was filtered to 0.45 μm to remove fine particles and was passed through an ultraviolet (UV) sterilizer.
- The well water is characterized as moderately-hard water. The specific conductance, hardness, alkalinity, and pH content of the well water during the approximate four-week period immediately preceding the test were presented in the full study report. The results of periodic analyses performed to measure the concentrations of selected organic and inorganic constituents in the well water were given in the full study report.

TEST APPARATUS
- A continuous-flow diluter was used to deliver well water to the treatment and control groups. The flow of dilution water to the test chambers was controlled by rotameters. The delivery of water from the rotameters was checked prior to the test and at approximately weekly intervals thereafter. Approximately 6 volume additions of test water were delivered to the test chambers every 24 hours. 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 test chambers used during both uptake and depuration phases 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 19.3 cm. Test chambers were positioned in a temperature-controlled water bath designed to maintain the target temperature throughout the test period. Aeration was added to each test chamber prior to the initiation of the uptake phase. 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.

PREPARATION OF THE DIET
- Bluegill were fed a commercially-prepared diet of Finfish Starter #2 Crumble supplied by Zeigler Brothers, Inc., Gardners, Pennsylvania. The results of periodic analyses performed to measure the concentrations of selected contaminants in the diet used by EAG Laboratories-Easton and the characterization of the test diet were presented in the full study report.
- The 500 μg/g diet mixture of the test item was prepared in the following manner: 1) 0.0492 g of the unlabeled test item was weighed into a small beaker; 2) 99.488 g of Zeigler Brothers Crumble #2 fish diet was weighed into a large weigh boat; 3) a small portion of the feed was then added to the beaker containing the unlabeled test item and mixed well with a metal spatula and then transferred back to the initial weight boat; 4) this process was then repeated several times, each time rinsing the small beaker with feed from the weigh boat until all of the unlabeled test substance had been transferred to the weigh boat; 5) the test item dosed feed was transferred from the weigh boat to a 16 oz. French square; 6) the [14C] labeled test item was then added to the mixture; 7) 0.500 mL of cod liver oil was added to the French square bottle and then mixed well using a vortex; 8) the fortified diet was placed on a shaker table that was set as approximately 250 rpm for approximately 1 hour with the bottle being rotated a quarter turn every 15 minutes.

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- The bluegill, Lepomis macrochirus, was selected as the test species for this study based on past use history in the laboratory. Bluegill used in the test were obtained from Osage Catfisheries, Inc., Osage Beach, Missouri and hatched on approximately June 10, 2016. Identification of the species was verified by the supplier.
- During the 14-day period preceding the test, water temperatures ranged from 22.0 to 23.1°C, measured with a hand-held liquid-in-glass thermometer. The pH of the water ranged from 8.2 to 8.4, measured with a Thermo Scientific Orion Benchtop 4 Star Plus pH/ISE meter. Dissolved oxygen ranged from 8.2 to 9.0 mg/L (≥94% of saturation), measured with a Thermo Scientific Orion Benchtop 3 Star Plus dissolved oxygen meter or equivalent. All fish in the culture during this time appeared normal and showed no signs of disease or stress. At test initiation, the bluegill were collected from the holding tanks and impartially distributed two at a time to the test chambers until each contained 70 fish. During acclimation and depuration phases, bluegill were fed a commercially-prepared diet of Finfish Starter #2 Crumble supplied by Zeigler Brothers, Inc., Gardners, Pennsylvania. The same diet was used to prepare the test diet that was used to feed the bluegill during the uptake phase throughout the study. Fish were not fed on the last day of the study so that gut contents could be purged.

LOADING
- Biomass loading rates were calculated using the wet weights of a subsample of fish collected from the lot used during the study since this was considered to be representative of the biomass in test chambers during the test. The average total length and weight of fish sampled on Day 0 of the uptake phase was 4.4 ± 0.50 cm (3.5 – 5.0 cm) and 1.10 ± 0.397 g (0.51 – 1.61 g), respectively.
- 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 0.1529 g fish/L/day. Instantaneous loading (the total wet weight of fish per liter of water in the tank) was 0.9632 g/L.

Route of exposure:

feed

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

14 d

Total depuration duration:

12 d

Hardness:

Measurements of hardness and alkalinity were typical of EAG Laboratories-Easton well water (see Table 12, attached)

Test temperature:

22.0 to 22.8 °C

pH:

8.1 to 8.4

Dissolved oxygen:

≥ 8.1 mg/L (93% of saturation)

Salinity:

Not applicable

Conductivity:

Measurements of conductivity were typical of EAG Laboratories-Easton well water (see Table 12, attached)

Details on test conditions:

PRELIMINARY TRIAL
- A non-GLP pilot study was conducted to evaluate the biomagnification potential of test item in the bluegill (Lepomis macrochirus) and presented in the full study report The pilot study was conducted with a nominal diet concentration of 500 μg/g test item.
- The procedures used to prepare the fortified test diet for the definitive study were established during the pilot study. The stability, homogeneity and storage conditions of the test substance in the treated diet were confirmed during the pilot study. A leaching trial was also conducted during the pilot study and results indicated that no test substance leached from the treated diet after approximately 3 minutes of exposure in the dilution water.
- The results of the pilot study showed that there was minimal uptake of test item into the fish tissue, BMF value was estimated to be 0.0038. In addition, analysis of the gut tract tissue indicated that most of the test substance remained in the gut and was not absorbed into the fish tissue.

TEST INITITIATION
- At test initiation, the bluegill were collected from the acclimation tanks and impartially distributed, using dip nets, two at a time to the test chambers until the test chambers contained 70 fish.
- When handling of the fish occurred it was done carefully, gently and quickly.

BIOLOGICAL OBSERVATIONS
- All fish were observed daily to evaluate the number of mortalities and the number of individuals exhibiting signs of abnormal behavior.

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 906 lux and was 1332 lux at the start of the depuration phase. Light intensity was measured using a SPER Scientific Model 840006 light meter.
- The target test temperature during the test was 22 ± 1°C. Temperature was measured in each test chamber at the beginning and end of the uptake and depuration phases test and at approximately weekly intervals during each phase of the test using a digital thermometer. Temperature also was monitored continuously in the negative control test chamber using a validated environmental monitoring system (AmegaView Central Monitoring System). The system measurements were calibrated prior to initiation of the uptake and depuration phases with a digital thermometer.
- Dissolved oxygen measurements were made in each test chamber daily during the uptake and depuration phases using a Thermo Scientific Orion Star A213 Benchtop RDO/DO meter. Measurements of pH were made in each test chamber at the beginning and end of the uptake and depuration phases and approximately weekly during the test using a Thermo Scientific Orion Dual Star pH/ISE meter.
- Hardness, alkalinity and conductivity were measured in the control and treatment groups at the beginning and end of the uptake and depuration phases, 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. Conductivity was measured using a Thermo Scientific Orion Star A122 Portable Conductivity meter.

FEEDING OF FISH
- During the uptake phase, the fish were fed the appropriate test substance or control diet daily at approximately 2% of body weight (wet weight). The initial feed rate was based on weight measurements of a sample of fish from the stock population collected on Day 0. The amount of feed was adjusted based on the wet weights of sampled fish at each interval during the testing period to account for growth during the test. Any uneaten food (as well as feces) were removed from the test chambers shortly after feeding. Fish were not fed on the last day of the study so that gut contents could be purged.

Nominal and measured concentrations:

- Test item 500 μg/g (nominal)
- Test item 479 μg/g (mean measured as total radioactivity)

Reference substance (positive control):

no

Details on estimation of bioconcentration:

DATA ANALYSES
- The dietary exposure bioaccumulation test allows for the determination of the substance-specific half-life (t1/2, from the depuration rate constant, k2), the assimilation efficiency (absorption across the gut; α), the kinetic biomagnification factor (BMFK), the lipid-corrected kinetic biomagnification factor (BMFKL) and the growth and lipid-corrected kinetic biomagnification factor (BMFKgL) for the test substance from fish.
- The depuration rate constants and biomagnification factors listed below, including 95% confidence intervals or standard deviations when possible, were estimated using methods outlined in the OECD Guideline 305:
(i) kg (growth rate constant; day-1)
(ii) k2 (overall depuration rate constant; day-1)
(iii) k2g (growth-corrected depuration rate constant; day-1)
(iv) C0,m (measured time 0 concentration, the concentration in fish at end of uptake; μg/g)
(v) C0,d (derived time 0 concentration of depuration phase; μg/g)
(vi) Ig (effective feeding rate, adjusted for growth; g food/g fish/day)
(vii) Cfood (chemical concentration in the food; μg/g)
(viii) α (substance assimilation efficiency)
(ix) BMFK (kinetic dietary BMF)
(x) BMFKg (growth-corrected kinetic dietary BMF)
(xi) t1/2 (half-life in days)
(xii) t1/2g (growth-corrected half-life in days)
(xiii) LC (lipid correction factor)
(xiv) BMFKgL (lipid-corrected growth-corrected kinetic BMF)
- Statistical analysis and linear regressions used to estimate depuration rate constants were performed using Excel 2010.

Conc. / dose:

500 µg/g food

Temp.:

>= 22 - <= 22.8 °C

pH:

8

Type:

BMF

Value:

0.003 dimensionless

Basis:

other: whole fish

Calculation basis:

other: growth and lipid corrected

Remarks on result:

other: assimilation efficiency (α) 0.00526

Rate constant:

other: depuration rate constant

Value:

0.171

Remarks on result:

other: K2

Details on results:

PHYSICAL AND CHEMICAL MEASUREMENTS OF WATER
- Water temperatures in the test chambers ranged from 22.0 to 22.8 °C, and both manual and continuous temperature measurements were within the range of 22 ± 1°C established for the test (see Table 12, attached). Measurements of water pH ranged from 8.1 to 8.4 during the test (see Table 12, attached). Dissolved oxygen remained ≥ 8.1 mg/L (93% of saturation) throughout the test (see Table 12, attached). Measurements of hardness, alkalinity and conductivity were typical of EAG Laboratories-Easton well water (see Table 12, attached).
- 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.

OBSERVATIONS OF MORTALITY AND CLINICAL SIGNS OF TOXICITY
- There were no observed mortalities in the negative control or treatment group during the test. All fish appeared normal and healthy throughout the test.

RADIOLABELED ANALYSIS OF TEST SUBSTANCE
- Aliquots of the [14C] labeled test item diluted in acetonitrile were transferred to scintillation vials containing 10 mL of Ultima Gold XR scintillation cocktail, and the solutions were analyzed by liquid scintillation counting (LSC). The mean measured concentration of the [14C] labeled test item was 10.2 mg/mL.

BACKGROUND RADIOACTIVITY
- In addition to the test samples, a combined sample of 10 mL of Permafluor E+ and 8 mL of Carbo-Sorb E were analyzed at each interval to determine background radioactivity. The background contribution for each interval and matrix type was automatically subtracted by the LSC’s software.

CONCENTRATIONS OF TEST ITEM IN DIET
- The concentrations of test item in the test diet collected prior to exposure are presented in Table 1 (attached). The concentrations of test item in test diet collected 2 (Day 0 Uptake) and 198 days after preparation are presented in Table 2 (attached). As the diet was inadvertently not sampled on Day 14 of the Uptake phase, diet was sampled approximately 6 months after the termination of the in-life portion of the study (198 days after preparation of diet). The mean measured concentration of test item in the test diet was 479 μg/g.

CONCENTRATIONS OF 14C-LABELED TEST ITEM IN FISH TISSUES
- The test item equivalent concentrations in tissues of fish exposed to a diet with a mean measured test concentration of 479 μg/g are presented in Tables 3, 4, and 5. The mean measured tissue concentration of test item in whole fish tissue at Day 14 of uptake was 3.10 μg/g (see Table 5, attached). The test item equivalent concentrations in gut and somatic (non-gut) tissue collected on Day 14 of uptake are presented in Tables 3 and 4 (attached), respectively. The mean measured test item equivalent concentrations in whole fish tissue (gut tract removed) was 1.15 μg/g. The results from this analysis indicate that a large portion of the test substance was retained in the gut tract.

DEPURATION RATE AND DERIVED TIME ZERO
- The depuration rate constant (k2) was calculated by plotting the natural logarithm of mean measured concentrations of the test substance (ln(concentration)) for the depuration period versus time (day). A linear least squares correlation was calculated for the ln(concentration) vs. time (day) data. The slope of the line was reported as the overall depuration constant (k2) and the intercept of the line was reported as the natural logarithm of the derived time zero concentration (C0,d). The overall depuration rate constant (k2) and the derived time zero concentration (C0,d) for the treatment group is presented in Table 7 (attached).
- In the test item treatment group the k2 value for whole fish tissue was 0.171. The derived time zero concentration (C0,d) for whole fish tissues in the test item treatment group was 0.268 μg/g. The natural logarithm transformed mean measured concentrations of test item and linear regression during the depuration phase is graphically represented in Figure 1 (attached).

GROWTH RATE
- The growth rate constants for each phase in the test item treatment group are presented in Table 8 (attached). Growth measurements (length and total wet weight) for individual fish collected during uptake and depuration were presented in the full study report. Growth rate constants (kg), were calculated by performing a linear least squares regression on the individual data of the control and treatment groups. The slopes of the linear regression were compared statistically using the student’s t-test (α = 0.05).
- There were no statistical differences between the slopes of the uptake and depuration phase growth data in the treatment group (p > 0.05), therefore the uptake and depuration data was pooled. There was no statistical difference between the overall slope of the treatment group in comparison to the control group (p > 0.05), therefore the control and treatment growth data were pooled to calculate an overall fish growth rate constant. The growth rate constant (kg) for the treatment group was 0.0233. The growth data and linear regression for the pooled control and treatment groups are graphically represented in Figure 2 (attached).

FOOD INGESTION RATE
- The food ingestion rate constant (I) was calculated using the desired amount of food to be fed per day, weight of fish and number of fish remaining in each tank. Feeding rates were adjusted based on the wet weights of sampled fish at each interval during the study. The food ingestion rate constant (I) was calculated to be 0.02 g food g-1 fish day-1.

ASSIMILATION EFFICIENCY
- The assimilation efficiency (α) is the efficiency of absorption of the test substance across the gut. The equations and parameters used to calculate the assimilation efficiency were presented in the full study report. The assimilation efficiency for whole fish tissues in the treatment group is presented in Table 9 (attached). The assimilation efficiency for whole fish tissue in the test item treatment group was 0.00526.

LIPID CONTENT
- Mean measured percent lipids in the treatment diet and fish tissue are presented in Table 10 (attached). The individual results from lipid analysis of diet and tissue were presented in the full study report. The mean measured percent lipid content in the control and treatment diet from samples collected on Day 0 of the uptake were 16.7 and 16.9%, respectively. The mean measured percent lipid content in the control and treatment diet from samples collected on Day 14 of uptake were 16.0 and 17.1%, respectively. The lipid content in all diets remained consistent over the course of the study.
- The mean measured lipid content in the whole fish tissue from the fish sampled from the control group on Days 0 and 14 of uptake and Day 12 of depuration was 6.23, 3.31, and 5.33%, respectively. The mean measured lipid content in the whole fish tissue from the fish sampled from the test item treatment group on Days 0 and 14 of uptake and Day 12 of depuration was 2.87, 2.85, and 4.67%, respectively.

LIPID CORRECTION
- The lipid correction (Lc) factor for the treatment group is presented in Table 10 (attached). The correction was calculated using the mean lipid fractions from the fish (Lfish) from Day 14 uptake and Day 12 depuration and the mean lipid fractions of diet (Lfood) analyzed on Days 0 and 14 of uptake. The lipid-correction factor was calculated to be 0.22 for the treatment group.

BMF CALCULATIONS
- The equations used to calculate the BMF values for the treatment group were presented in the full study report and results are presented in Table 11 (attached). The growth and lipid corrected BMF (BMFKgL) value for whole fish tissue of the test item treatment group was 0.0032. The time to reach 95% steady state (BMFSS) was 20 days and the growth corrected half-life (t1/2g) was 4.7 days.

CONDITIONS FOR VALIDITY OF THE TEST
- The criteria were used to judge the validity of the test and were met unless otherwise indicated:
(i) There was no mortality or adverse effects in the control or treatment groups.
(ii) Water did not differ by more than 2 °C in treatment or control groups.
(iii) The dissolved oxygen concentration was at least 60% of the air-saturation value throughout the majority of the exposure period.
(iv) The concentration of the test substance in fish diet before and at the end of uptake phase was within ± 20%.
(v) The homogeneity of the test substance in fish diet did not vary more than ± 15% from the mean.
(vi) Test substance was not detected control fish tissue.

Validity criteria fulfilled:

yes

Conclusions:

Bluegill were exposed to a control and treatment feed for 14 days. Mean measured concentration of the treatment diet was 497 μg/g test item. The growth and lipid corrected BMF value was 0.0032 in analytically determined whole fish tissues in the treatment group. The measured time zero concentration (tissue concentration at Day 14 uptake) in whole fish tissue was 3.10 μg/g, while the derived time zero concentration in whole fish tissue was 0.268 μg/g suggesting the presence of undigested food in the gut tract. When whole fish tissues were analyzed without the gut tract, the mean measured concentration in fish tissue was 1.15 μg/g.

Executive summary:

GUIDELINE

The objective of this study was to obtain laboratory data characterizing the bioaccumulation potential of test item in the bluegill, Lepomis macrochirus. The protocol was based on procedures outlined in OECD Guidelines for Testing of Chemicals, Guideline 305:Bioaccumulation in Fish: Aqueous and Dietary Exposure.As the BMF is a comparison of the concentration of a substance in an organism with that in the organism’s food, lipid is taken into account by correcting for the contents of lipid in the organism and in the food.

METHODS

The test was divided into two phases: uptake and depuration. During the uptake phase, bluegill were exposed an isotopic mixture of test item in diet at a sub-lethal concentration. The bluegill in the control group were exposed to an untreated diet. The nominal concentration of 500 μg/g test item was selected in consultation with the Sponsor. Each group consisted of one test chamber with 70 fish in each chamber. During the depuration phase, fish were exposed to an untreated diet only. The duration of the uptake phase was 14 days and the depuration phase was 12 days. During both phases of the test, test organisms and water samples were collected and analyzed for 14C radioactivity. These values were used to determine the growth-corrected substance-specific half-life (t1/2g, from the growth-corrected elimination rate constant,k2g), the assimilation efficiency (absorption across the gut; α), the kinetic biomagnification factor (BMFK) and the lipid-corrected kinetic biomagnification factor (BMFKL) for whole fish tissues.

RESULTS

Bluegill were exposed to a control and treatment feed for 14 days. Mean measured concentration of the treatment diet was 497 μg/g test item. The growth and lipid corrected BMF value was 0.0032 in analytically determined whole fish tissues in the treatment group. The measured time zero concentration (tissue concentration at Day 14 uptake) in whole fish tissue was 3.10 μg/g, while the derived time zero concentration in whole fish tissue was 0.268 μg/g suggesting the presence of undigested food in the gut tract. When whole fish tissues were analyzed without the gut tract, the mean measured concentration in fish tissue was 1.15 μg/g.

Reference 2

Endpoint:

bioaccumulation in sediment species: invertebrate

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 August 2017 to 25 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 315 (Bioaccumulation in Sediment-dwelling Benthic Oligochaetes)

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Details on sampling:

ANALYTICAL SAMPLING
- Sediment and overlying water samples were collected from three test chambers of the solvent
control and the 100 mg/kg treatment group on Days 0, 1, 3, 7, 14, 21 and 28 of the uptake phase and on Days 0, 1, 3, 5, 7 and 10 of the elimination phase. The replicates used for sample collection on Day 0 of the uptake phase did not contain oligochaetes, whereas at test initiation, oligochaetes were placed in all other replicates.
- Tissue samples were collected on Days 1, 3, 7, 14, 21 and 28 of the uptake phase and on Days 0, 1, 3, 5, 7 and 10 of the elimination phase. Although Day 28 of the uptake phase and Day 0 of the elimination phase are on the same day, separate samples were analyzed for each interval. Prior to sampling the Day 0 elimination tissue samples, the organisms were first transferred from treated sediment to untreated sediment and an amount of time was allowed to pass (approximately 4 hours) before collecting the tissue for analysis. Tissue samples were also collected on Day 28 of the uptake phase from the negative control for the determination of lipid content. Tissue samples were collected by sieving the sediment to remove organisms. The organisms were rinsed free of as much sediment as possible and placed in a tared liquid scintillation vial and a wet weight was obtained. Tissue samples were analysed immediately or stored frozen until analysis could be performed.

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

EXPERIMENTAL DESIGN
- The test was divided into two phases: the uptake (exposure) phase and the elimination
(post-exposure) phase. During the uptake phase, oligochaetes were exposed to one sub-lethal test
concentration and a negative and solvent control. The nominal test concentration selected in consultation with the sponsor was 100 mg the test item mg/kg of sediment based on the dry weight of the sediment. Oligochaetes in the solvent control group were exposed under identical conditions without test substance, but with the same amount of solvent used in the treatment group. Oligochaetes in the negative control group were exposed without test substance or solvent. Each test chamber contained the same quantities of sediment and overlying water. Thirty-six replicate test chambers were prepared for both the treatment group and the solvent control group for exposure of the test organisms (i.e. 18 replicates for sampling during the uptake phase plus 18 replicates for sampling during the elimination phase). An additional three replicates were prepared without organisms for the treatment group and the solvent control group for analytical sampling on Day 0. Four replicate test chambers were prepared for the negative control group for sampling at the end of the uptake phase.
- The results of the study are based on the mean measured test concentrations in the sediment during the uptake phase. The duration of the uptake phase typically will vary according to the time required to reach steady-state, but was not to exceed 28 days. Sediment and overlying water samples were collected and analyzed from three sacrificed replicates of the treatment group and solvent control group on Days 0, 1, 3, 7, 14, 21 and 28 of the uptake phase. Results of the analyses in the sediment were used to verify the exposure over time. Additionally, sediment and overlying water samples were collected on Days 0, 1, 3, 5, 7 and 10 of the elimination phase. The duration of the elimination phase typically will vary according to the time required to reach 10% of the concentration measured in tissues at the end of the uptake phase, but was not to exceed 10 days. Worm tissue samples were collected from the culture on Day 0 and from three replicates from the treatment group and solvent control group on days 1, 3, 7, 14, 21 and 28 of the uptake phase and on days 0, 1, 3, 5, 7 and 10 of the elimination phase. An additional four replicates from the negative control group were sacrificed at the end of the uptake phase for the determination of lipid content in the tissue. The tissue concentrations were used to calculate the uptake rate constant (ks), the elimination rate constant (ke), and the kinetic bioaccumulation factor (BAFK). The bioaccumulation factor (BAF) was calculated, based on the concentration of the test item in the oligochaetes compared to the concentration of the test item in the sediment. Additionally, the residue level in the oligochaetes at the end of the elimination phase (non-eliminated residue; NER) was also determined.

TEST SEDIMENT
- Formulated sediment based on the recommendations of OECD Guideline 315 (1) was used as the test sediment. The sediment was composed of approximately 5% sphagnum peat moss, 20% silt and clay (kaolin clay) and 75% industrial quartz sand. The dry constituents of the sediment were mixed in a PK Twinshell mixer for 40 minutes, and the batch was stored under ambient conditions until used. The pH of the dry sediment was determined prior to use in the test and was within the desired range of 7.0 (± 0.5). The final pH of a 1:1 ratio of the sediment and water was 7.5. A sample of formulated
sediment similar to that which was used in the test was sent to Agvise Laboratories, Northwood, North Dakota, for characterization and analysis of total organic carbon (TOC), and a summary of the sediment characterization was presented in the full study report. The percent organic carbon of the sediment was determined to be 1.5%. The composition of the sediment, as determined by Agvise Laboratories, was 76% sand and 24% silt and clay. The results of periodic analyses performed to measure the concentrations of selected organic and inorganic constituents in a representative sample of formulated sediment like that used in the test were presented in the full study report. The sediment had a percent moisture content of 13.7% just prior to use in the uptake phase and 13.9% just prior to the elimination phase.

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

TEST ORGANISM
- The oligochaete, Lumbriculus variegatus, was selected as the test species for this study. Oligochaetes are representative of an important group of sediment-dwelling invertebrates and were selected for use in the study based upon past history of use and ease of culturing in the laboratory. The organisms used in the test were obtained from cultures maintained at EAG Laboratories; however, the original culture was received from Aquatic Research Organisms, Hampton, New Hampshire. The identity of the species was verified by the supplier.
- The organisms were held in aquaria containing water from the same source as the water used during the test and a layer of clean sand. During the 14-day holding period immediately preceding the test, water temperature ranged from 17.8 to 19.3 °C, while the pH ranged from 8.4 to 8.6 and dissolved oxygen ranged from 8.6 to 9.1 mg/L.
- During holding, the oligochaetes appeared normal. At test initiation, synchronized adult oligochaetes were selected from the culture and sequentially added one and two at a time to transfer containers until each transfer container contained 50 individuals. Only intact, complete worms of similar size, which were actively swimming or crawling upon gentle mechanical stimulus, were selected. Transfer containers were impartially assigned to test chambers. All transfers were made below the air/water interface using a wide-bore pipette. Four replicate test chambers in the negative control and 36 replicates in the solvent control and 100 mg/kg treatment group contained organisms, while three test chambers in the solvent control and 100 mg/kg treatment group were maintained without organisms for day 0 analytical purposes.
- Oligochaetes were fed wheat grass during the holding period and nettle leaf powder (Urtica sp.) during the test. The food used during the test was incorporated into the sediment at a rate of 0.4% of the dry weight of sediment. During the uptake phase, the food was incorporated into the sediment during the dosing procedure. During the elimination phase, the food was incorporated into the sediment during the sediment preparation procedure. No further addition of food was made during the test. There was no evidence of fungal growth or excess food on the sediment surface during the test.

Route of exposure:

sediment

Water / sediment media type:

artificial sediment

Total exposure / uptake duration:

28 d

Total depuration duration:

10 d

Hardness:

Measurements of hardness, alkalinity, and ammonia were typical for EAG Laboratories well water (see Table 11, attached)

Test temperature:

20 ± 2 °C

pH:

8.4 to 8.7

Dissolved oxygen:

≥90% (8.1 mg/L) of saturation throughout the test.

Salinity:

Not applicable

Conductivity:

Measurements of conductivity were typical for EAG Laboratories well water (see Table 11, attached)

Details on test conditions:

TEST WATER
- The water used for holding and testing was freshwater obtained from a well approximately 40 meters deep located on the EAG Laboratories site. 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 where the water was aerated with spray nozzles. Prior to use in the test system, the water was filtered to 0.45 µm to remove fine particles and was UV-sterilized.
- The well water is characterized as moderately-hard water. The specific conductance, hardness, alkalinity, pH and total organic carbon (TOC) content of the well water during the approximate four-week period immediately preceding the test were presented in the full study report. The results of periodic analyses performed to measure the concentrations of selected organic and inorganic constituents in the well water were presented in the full study report.

TEST APPARATUS
- The study was conducted in a temperature-controlled room. Each test chamber was a 2-L glass
beaker, holding 400 grams of dosed or un-dosed sediment and 1600 mL of test water. The sediment :water ratio was 1 : 4. The depth of sediment in a representative test chamber (negative control replicate A) at the start of the test was 2 cm. The depth of overlying water in negative control replicate A was 13 cm at the start of the test. Loose plastic covers were placed over the test chambers for the duration of the study. Approximately two times per week (e.g., Tuesday, Thursday), a portion of the overlying water (approximately 800 mL) was removed from each test chamber and replaced with clean test water. Aeration was applied to each test chamber through a glass pipette that extended to a depth not closer than 2 cm from the sediment surface. In order to maintain a dissolved oxygen concentration ≥40% of air saturation, air was bubbled into each test chamber at a rate greater than 1 bubble/second but not so great as to disturb the sediment. Test chambers were arranged in an unbiased order and were labeled with the project number, test concentration and replicate. The general operation of the test apparatus was checked visually at least once each day during the test.

PREPARATION OF TEST CONCENTRATIONS
- The test substance was administered to the test organism in sediment. This route of administration was selected because it represents the most likely route of exposure to sediment dwelling organisms.
- The concentration of the test substance were prepared on a dry weight basis, (i.e., mg test substance/kg dry sediment). The test concentration was not adjusted for the active ingredient of the test substance during preparation, and is based on the test substance as received. The nominal test concentration was 100 mg/kg. The radiolabeled test substance (13526) was received as a stock solution prepared in acetonitrile at a nominal concentration of 11.5 mg/mL (specific activity of 188351 dpm/µg). The radioactivity of the radiolabeled stock was confirmed prior to use in the study by analyzing three, 100 µL samples of stock solution by liquid scintillation counting (LSC). The measured concentration of the primary radiolabeled stock was 10.5 mg/mL, or 91.6% of the expected radioactivity.
- A primary stock solution of non-radiolabeled test substance was prepared by mixing 2.4000 g of test substance into 200 mL of HPLC-grade acetone at a nominal concentration of 12 mg/mL. The dosing stock solution was prepared as an isotopic dilution at a nominal concentration of 10 mg/mL. A 0.334-mL aliquot of the radiolabeled primary stock (equivalent to 3.507 mg of test substance) was added to a 500-mL glass graduated cylinder. A 137.208-mL aliquot of the non-radiolabeled primary stock (equivalent to 1646.5 mg of test substance) was also added to the 500-mL glass graduated cylinder. The cylinder was brought to a final volume of 165 mL with acetone and inverted to mix. The resulting specific activity of the dosing stock was 400 dpm/µg. The stock appeared clear and colorless with no visible precipitates after mixing.
- To prepare the dosed sediment for the treatment level, an 18-mL volume of the dosing stock solution was added to 90.0 g of sand in a labeled glass beaker and was mixed by hand with a glass stir rod until homogenous. This dosed “sand premix” was placed under a fume hood and the acetone was allowed to evaporate for approximately 2 hours. The 90 g sand premix was added to 810 g dry weight (938.6 g wet weight) of untreated formulated sediment in a 2000-mL plastic Nalgene® bottle and mixed on a rotary mixer for approximately 2 hours. An additional 900 g dry weight (1042.9 g wet weight) of untreated formulated sediment was added to the premix to achieve a final dry weight of 1800 g. Just prior to an overnight mix, a pre-weighed aliquot of nettle leaf powder (7.2 g) was added to each of the Nalgene bottles. This amount of food was equivalent to 0.4% of the dry weight of sediment and was added to the batch sediment on the day it was dosed to ensure the food was in sufficient contact with the test substance. The 1800 g batch of sediment and food was mixed on a rotary mixer for approximately 39 hours prior to transfer of the dosed sediment to the test chambers. Since a solvent (acetone) was used in the preparation of the test sediments, a solvent control was included in the test design. The solvent control sediment was prepared using 18 mL of acetone, with the same mixing procedures as the treated sediment but with no test substance added. The negative control sediment was prepared without the addition of test substance or solvent. A total of nine 1800 g batches of sediment were prepared for both the treatment group and the solvent control group for the uptake phase using the procedure stated above. A single 1800 g batch of sediment was prepared for the negative control group for the uptake phase.
- Since the organisms from the solvent control and 100 mg/kg treatment group were transferred to un-dosed sediment for the elimination phase, four days prior to the elimination phase (Day 24 of the test), nine 1800 gram batches of sediment were prepared without the addition of test substance or solvent. 90.0 g of sand was added to 810 g dry weight (940.8 g wet weight) of untreated formulated sediment in a 2000 mL plastic Nalgene bottle and mixed on a rotary mixer for one hour. Next, an additional 900 g dry weight (1045.3 g wet weight) of untreated formulated sediment was added to the bottle and the final weight of 1800 grams was mixed on a rotary mixer for approximately 45 hours. Just prior to the overnight mix, a pre-weighed aliquot of nettle leaf powder (7.2 g) was added to each of the Nalgene bottles. This amount of food was equivalent to 0.4% of the dry weight of sediment and was added to the batch sediment on the day it was prepared to be consistent with the sediment preparation for the uptake phase. For both the uptake and the elimination phases, test chambers were prepared and allowed to equilibrate for 2 days prior to adding organisms.
- During the uptake phase, four replicate test chambers were prepared for the negative control and 39 replicate test chambers were prepared for both the solvent control group and the 100 mg/kg treatment group. For each replicate, 400 g of sediment was placed in a 2-L glass beaker. Then, 1600 mL of overlying water (well water) was poured into the chamber over a baffle to minimize the disturbance to the sediment. Test chambers were indiscriminately positioned in an environmentally controlled room approximately 2 days prior to test initiation to condition the sediment prior to introduction of organisms. At the beginning of the test, the overlying water in all test chambers appeared clear and tan.
- Eighteen replicate test chambers were prepared for the solvent control group and the 100 mg/kg treatment group for use in the elimination phase. For each replicate, 400 grams of untreated sediment was placed in 2-L glass beakers. Next, 1600 mL of overlying water (well water) was slowly added to each test chamber by pouring over a baffle to minimize the disturbance to the sediment. Gentle aeration was maintained in each test chamber during both the uptake and elimination phases using a glass pipette that extended no closer than 2-cm from the surface of the sediment. Aeration was supplied at a rate of greater than one bubble per second, but not so great as to disturb the sediment. Test chambers were indiscriminately positioned in an environmentally controlled room approximately 2 days prior to initiation of the elimination phase to condition the sediment prior to introduction of organisms. At the end of the test, the overlying water in all test chambers appeared cloudy and light tan.

ENVIRONMENTAL CONDITIONS
- The test system was illuminated using fluorescent tubes that emit wavelengths similar to natural sunlight. The lights were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in light intensity. Light intensity was measured at the water surface of one representative test compartment (Negative Control replicate A) at the beginning of the test using a SPER Scientific Model 840006 light meter and found to be 671 lux.
- The target test temperature during the study was 20 ± 2 °C. Temperature was measured in the overlying water of one alternating replicate test chamber from each treatment and control group daily during the test using a hand-held digital thermometer. Temperature also was measured continuously in a beaker of water adjacent to the test chambers using a validated environmental monitoring system (AmegaView Central Monitoring System). The system measurements were verified prior to exposure initiation with a
digital thermometer.
- Dissolved oxygen measurements were made on samples of overlying water collected from one
alternating replicate test chamber of each treatment and control group daily during the test. Measurements of pH were made on samples of overlying water collected from one alternating replicate test chamber of each treatment and control group at test initiation, once each week during the test, and at test termination. Hardness, alkalinity, specific conductance and ammonia were measured in a sample of overlying water collected from one alternating replicate test chamber from each treatment and control group at the beginning and end of both the uptake and elimination phases.
- Dissolved oxygen was measured using a Thermo Scientific Orion Star A213 Benchtop RDO/DO meter and measurements of pH were made using a Thermo Scientific Orion Dual Star pH/ISE Meter. Hardness and alkalinity measurements were made by titration. Specific conductance was measured using a Thermo Scientific Orion Star A122 portable conductivity meter. Ammonia was measured using a Thermo Scientific Orion Dual Star pH/ISE meter.

PROCEDURES AND BIOLOGICAL OBSERVATIONS AND MEASUREMENTS
- In addition to the organisms placed in the test chambers at the beginning of the test, an additional 50 organisms were impartially selected from the culture at the beginning of the test and measured for dry weight. The weight of this subset of organisms was used to determine the ratio of organic carbon in the sediment to the dry weight of organisms. The test chambers were observed daily to make visual assessments of abnormal behavior (e.g. leaving the sediment, climbing the walls of the test compartment). The test was divided into two phases: uptake and elimination. The duration of the uptake phase was 28 days, and the elimination phase was 10 days.
- At each sampling interval during both the uptake and elimination phases, after collection of overlying water and sediment samples, the organisms in each of the three selected analytical replicates in the treatment and solvent control groups were segregated from the sediment by sieving, and the number and wet weight of surviving organisms was determined. At the end of the uptake phase, oligochaetes in the remaining uptake phase replicates from the solvent control and 100 mg/kg treatment group were transferred to sediment-water systems free of test substance for the elimination phase. Also at the end of the uptake phase, oligochaetes in the four replicates from the negative control were processed for lipid analysis and did not continue on into the elimination phase. As in the uptake phase, test chambers in the elimination phase were observed daily and visual assessments of any abnormal behavior was recorded.

Nominal and measured concentrations:

- Test item 100 mg/kg (nominal)
- Test item 92.1 mg/kg (measured in sediment during uptake)

Reference substance (positive control):

no

Details on estimation of bioconcentration:

DATA ANALYSES
- The equations used for BAF (bioaccumulation factor) and BAFK (kinetic bioaccumulation factor)
calculations are described in OECD Guideline 315 (1). The kinetic uptake rate (ks) and elimination rate (ke) were calculated for oligochaete tissue and then used to calculate a kinetic bioaccumulation factor (BAFK = ks/ ke). A non-eliminated residues (NER) level in oligochaetes was also calculated. The normalized BAF is also expressed as a function of the lipid content of the oligochaetes and of the total organic carbon (TOC) of the sediment.

Conc. / dose:

92.1 mg/kg sediment dw

Temp.:

20 °C

pH:

8.55

Type:

BAF

Value:

6.4 dimensionless

Basis:

other: mean tissue concentration

Calculation basis:

other: not based on steady state concentrations

Remarks:

calculated as mean measured Day 28 tissue concentration divided by mean measured Day 28 sediment concentration

Remarks on result:

other: Day 28 BAF

Conc. / dose:

92.1 mg/kg sediment dw

Temp.:

20 °C

pH:

8.55

Type:

BAF

Value:

8.64 dimensionless

Basis:

other: mean tissue concentration

Calculation basis:

other: not based on steady state concentrations

Remarks:

calculated as mean measured Day 21 tissue concentration divided by mean measured Day 21 sediment concentration

Remarks on result:

other: Day 21 BAF

Remarks:

This can be used as a worst case scenario since the highest measured tissue concentrations occurred at the Day 21 interval

Conc. / dose:

92.1 mg/kg sediment dw

Temp.:

20 °C

pH:

8.55

Type:

BAF

Value:

4.42 dimensionless

Basis:

other: mean tissue concentration

Calculation basis:

other: (Day 28 uptake mean measured tissue concentration / lipid content) / (Day 28 uptake concentration in the sediment / organic carbon content)

Remarks on result:

other: Normalized BAF

Conc. / dose:

92.1 mg/kg sediment dw

Temp.:

20 °C

pH:

8.55

Type:

BAF

Value:

6.35 dimensionless

Basis:

other: kinetic bioaccumulation factor

Calculation basis:

other: uptake rate constant in tissue / elimination rate constant

Remarks on result:

other: BAFk

Elimination:

yes

Parameter:

other: NER

Remarks:

non-eliminated residue level

Remarks on result:

other: The non-eliminated residue level was calculated as a percentage from the ratio of the average concentration in the worms on Day 10 of the elimination phase and the average concentration in the worms at the end of the uptake phase.

Details on results:

MEASUREMENTS OF TEST CONCENTRATIONS IN SEDIMENT AND OVERLYING WATER
- Results of analyses to measure concentrations of the test item (based on total radioactive residues) in samples collected during the definitive test are presented in Tables 3 - 8. The nominal concentration selected for use in this definitive test was 100 mg/kg.
- Since it could not be demonstrated that equilibrium (steady-state) had been reached in the treatment group, the uptake phase lasted 28 days, the maximum allowed by the guideline. Samples of sediment and overlying water were collected from the solvent control and the test concentration on Days 0, 1, 3, 7, 14, 21 and 28 of the uptake phase. The purpose of these samples was to verify the concentrations in sediment at test initiation and to monitor the test substance in the sediment over the course of the study. Results are based on the mean measured concentration of total radioactive residues (TRR) in sediment for each sampling day during the uptake phase.
- Analytical results for solvent control sediment and overlying water samples analyzed on Days 0, 1, 3, 7, 14, 21 and 28 were all below the limit of quantitation (LOQ); (Tables 3 and 5). The mean measured concentration of the test item equivalents in sediment from the 100 mg/kg test concentration on Day 0 was 93.4 mg/kg (Table 3). Mean measured concentrations of the test item equivalents in sediment from the 100 mg/kg test concentration on Days 1, 3, 7, 14, 21 and 28 of the uptake phase were 101.1, 78.7, 95.3, 96.5, 93.0 and 86.7 mg/kg, respectively. The overall mean measured concentration of the test item equivalents in sediment for the 100 mg/kg treatment group during the uptake phase was 92.1 mg/kg, or 92.1% of nominal. Measured concentrations in the sediment during the elimination phase are presented in Table 4 (attached) and were all below the limit of quantitation (LOQ = 0.395 mg/kg).
- The measured concentration of the test item equivalents in overlying water from the 100 mg/kg test concentration ranged from 0.263 to 0.617 mg/L (Table 5) during the uptake phase. The mean measured concentration of the test item equivalents in overlying water from the 100 mg/kg test concentration on Day 0 was 0.447 mg/L. Mean measured concentrations of the test item equivalents in overlying water from the 100 mg/kg test concentration on Days 1, 3, 7, 14, 21 and 28 of the uptake phase were 0.514, 0.402, 0.571, 0.468, 0.532 and 0.319 mg/L, respectively. Measured concentrations in the overlying water during the elimination phase are presented in Table 6, and were all below the limit of quantitation (0.0125 mg/L).

MEASUREMENT OF TEST ITEM IN TISSUE
- Worm tissue samples were collected from the solvent control and the 100 mg/kg test concentration on Days 1, 3, 7, 14, 21 and 28 of the uptake phase and on Days 0, 1, 3, 5, 7 and 10 of the elimination phase. Measured concentrations in solvent control samples were below the limit of quantitation (LOQ = 1.25 mg/kg) at all sampling intervals except for Days 14 and 21 of the uptake phase. One sample from the Day 14 analysis and two samples from the Day 21 analysis had a measureable amount of radioactivity present in the solvent control but is due to suspected contamination of the samples, since radioactivity was not detected in the other solvent control tissue samples on the same sampling days or any other solvent control tissue samples for the remainder of the test. Additionally, the Day 14 solvent control tissue sample was reanalyzed and found to have no measureable radioactivity. The concentrations of the test item equivalents in tissues of oligochaetes exposed in the 100 mg/kg treatment group during the uptake and elimination phases of the study are presented in Tables 7 and 8 (attached), respectively.
- The Day 21 BAF and Day 28 BAF values for oligochaetes exposed to sediment incorporated the test item were 8.64 and 6.40 respectively (Table 10). The Day 21 BAF represents a worst case scenario since the highest measured tissue concentration occurred at the Day 21 interval. Steady state occurs
when: (i) a plot of the bioaccumulation factors at each sampling period against time is parallel to the time axis; (ii) three successive analyses of BAF made on samples taken at intervals of at least two days vary no more than ± 20% of each other; and (iii) there are no significant differences between the three sampling periods (based on statistical comparisons e.g. analysis of variance and regression analysis). The BAF was not based on steady state concentrations, since the three conditions were not met for steady state. Measured concentrations in tissue exceeded the measured concentrations in sediment during the uptake phase of the test (see Tables 3, 4, 7 and 8, attached). There was a steep increase in measured tissue concentrations between Days 1 and Day 21, then a decrease on Day 28 of the uptake phase (see Figures 1 and 2, attached). The concentration of the test item equivalents in oligochaetes declined slowly during the elimination phase (see Figure 2, attached). The mean measured concentration of the test item equivalents in oligochaete tissue from the 92.1 mg/kg treatment group was 193 mg/kg by Day 10 of the elimination phase (see Table 8, attached), which is approximately 65% of the mean tissue concentration at Day 28 of uptake (554.7 mg/kg). The non-eliminated residue content on Day 10 of the elimination phase was 34.9%. The uptake rate was 0.670 and the elimination rate was 0.105. The BAFK was 6.35 mg/kg.
- The lipid content from the organisms found in the four negative control replicates on Day 28 of the uptake phase was determined to be 2.17% (see Table 9, attached). The percent organic carbon in the sediment was 1.5%. The normalized Day 28 BAF, based on the lipid content and percent organic carbon in the sediment was 4.42 (see Table 10, attached).

OBSERVATIONS AND MEASUREMENTS
- Measurements of temperature, dissolved oxygen, and pH of the overlying water in the test chambers are summarized in Table 11 (attached). Temperatures were within the 20 ± 2 °C range established for the test. Dissolved oxygen concentrations were ≥90% (8.1 mg/L) of saturation throughout the test. Measurements of pH ranged from 8.4 to 8.7 during the test. Measurements of hardness, alkalinity, conductivity and ammonia are summarized in Table 11 (attached), and individual measurements are presented in the full study report were typical for EAG Laboratories well water.
- Survival of oligochaetes in individual replicates at each sampling interval are presented in the full study report. Worms in all replicates appeared normal during the test, with few observations of abnormal behavior (i.e., organisms observed on the surface of the sediment) in the treatment and control groups, and no treatment-related signs of toxicity. At test termination, there was an increase in the numbers of oligochaetes present in the test chambers from the number of worms originally placed in each replicate, indicating that reproduction had occurred.
- At the beginning of the test, 50 organisms from the culture were impartially selected and used for the determination of dry weight. It was determined that the average individual dry weight of those organisms was 0.37 mg. It was determined that the formulated sediment had an organic content of 1.5%; therefore, the ratio of organic carbon to the dry weight of organisms was 279.9 g organic carbon : 1 g of dry tissue.

CONDITIONS FOR THE VALIDITY OF THE TEST
- The following criteria were used to judge the validity of the test and were met for this test:
1. The cumulative mortality of the worms (controls and treatment) until the end of the test should not exceed 20% of the initial number. Cumulative mortality did not exceed 20% in the treatment group (or controls) at any point during the test.
2. It should be demonstrated that the worms burrowed in the sediment to allow for maximum exposure. In this test, the worms were burrowed in the sediment for maximum exposure.

Validity criteria fulfilled:

yes

Conclusions:

The Day 28 bioaccumulation factor (BAF) for oligochaetes (Lumbriculus variegatus) exposed to sediment spiked with the test item at a nominal concentration of 100 mg test substance/kg dry sediment (mean measured concentration during the uptake phase of 92.1 mg the test item equivalents/kg dry sediment) was 6.40. The accumulation in tissue was swift and the elimination was quick but variable. The non-eliminated residue content on Day 10 of the elimination phase was 34.9%. The uptake rate was 0.670 and the elimination rate was 0.105. The BAFK was 6.35 mg/kg. The lipid content from the organisms found in the four negative control replicates on Day 28 of the uptake phase was determined to be 2.17%. The percent organic carbon in the sediment was 1.5%. The normalized Day 28 BAF, based on the lipid content and percent organic carbon in the sediment was 4.42.

Executive summary:

GUIDELINE

The objective of this study was to determine the bioaccumulation potential of the test item in the oligochaete, Lumbriculus variegatus, through sediment exposure. The protocol was based upon the OECD Guidelines for Testing of Chemicals, Guideline 315: Bioaccumulation in Sediment-dwelling Benthic Oligochaetes

 

METHODS

An initial trial was conducted but was terminated early and repeated due to high mortality in the treatment group. The test concentration was lowered for the definitive test.

 

The test was divided into two phases: the uptake (exposure) phase and the elimination (post-exposure) phase. During the uptake phase, oligochaetes were exposed to one sub-lethal test concentration and a negative and solvent control. The nominal test concentration selected in consultation with the sponsor was 100 mg the test item mg/kg of sediment based on the dry weight of the sediment. Oligochaetes in the solvent control group were exposed under identical conditions without test substance, but with the same amount of solvent used in the treatment group. Oligochaetes in the negative control group were exposed without test substance or solvent. Each test chamber contained the same quantities of sediment and overlying water. Thirty-six replicate test chambers were prepared for both the treatment group and the solvent control group for exposure of the test organisms (i.e. 18 replicates for sampling during the uptake phase plus 18 replicates for sampling during the elimination phase). An additional three replicates were prepared without organisms for the treatment group and the solvent control group for analytical sampling on Day 0. Four replicate test chambers were prepared for the negative control group for sampling at the end of the uptake phase.

 

The results of the study are based on the mean measured test concentrations in the sediment during the uptake phase. The duration of the uptake phase typically will vary according to the time required to reach steady-state, but was not to exceed 28 days. Sediment and overlying water samples were collected and analyzed from three sacrificed replicates of the treatment group and solvent control group on Days 0, 1, 3, 7, 14, 21 and 28 of the uptake phase. Results of the analyses in the sediment were used to verify the exposure over time. Additionally, sediment and overlying water samples were collected on Days 0, 1, 3, 5, 7 and 10 of the elimination phase. The duration of the elimination phase typically will vary according to the time required to reach 10% of the concentration measured in tissues at the end of the uptake phase, but was not to exceed 10 days. Worm tissue samples were collected from the culture on Day 0 and from three replicates from the treatment group and solvent control group on days 1, 3, 7, 14, 21 and 28 of the uptake phase and on days 0, 1, 3, 5, 7 and 10 of the elimination phase. An additional four replicates from the negative control group were sacrificed at the end of the uptake phase for the determination of lipid content in the tissue. The tissue concentrations were used to calculate the uptake rate constant (ks), the elimination rate constant (ke), and the kinetic bioaccumulation factor (BAFK). The bioaccumulation factor (BAF) was calculated, based on the concentration of the test item in the oligochaetes compared to the concentration of the test item in the sediment. Additionally, the residue level in the oligochaetes at the end of the elimination phase (non-eliminated residue; NER) was also determined.

 

RESULTS

The Day 28 bioaccumulation factor (BAF) for oligochaetes (Lumbriculus variegatus) exposed to sediment spiked with the test item at a nominal concentration of 100 mg test substance/kg dry sediment (mean measured concentration during the uptake phase of 92.1 mg the test item equivalents/kg dry sediment) was 6.40. The accumulation in tissue was swift and the elimination was quick but variable. The non-eliminated residue content on Day 10 of the elimination phase was 34.9%. The uptake rate was 0.670 and the elimination rate was 0.105. The BAFK was 6.35 mg/kg. The lipid content from the organisms found in the four negative control replicates on Day 28 of the uptake phase was determined to be 2.17%. The percent organic carbon in the sediment was 1.5%. The normalized Day 28 BAF, based on the lipid content and percent organic carbon in the sediment was 4.42.

Description of key information

Dietary exposure of fish

Bluegill were exposed to a control and treatment feed for 14 days. Mean measured concentration of the treatment diet was 497 μg/g test item. The growth and lipid corrected BMF value was 0.0032 in analytically determined whole fish tissues in the treatment group. The measured time zero concentration (tissue concentration at Day 14 uptake) in whole fish tissue was 3.10 μg/g, while the derived time zero concentration in whole fish tissue was 0.268 μg/g suggesting the presence of undigested food in the gut tract. When whole fish tissues were analyzed without the gut tract, the mean measured concentration in fish tissue was 1.15 μg/g (OECD 305).

Exposure of sediment organisms

The Day 28 bioaccumulation factor (BAF) for oligochaetes (Lumbriculus variegatus) exposed to sediment spiked with the test item at a nominal concentration of 100 mg test substance/kg dry sediment (mean measured concentration during the uptake phase of 92.1 mg the test item equivalents/kg dry sediment) was 6.40. The accumulation in tissue was swift and the elimination was quick but variable. The non-eliminated residue content on Day 10 of the elimination phase was 34.9%. The uptake rate was 0.670 and the elimination rate was 0.105. The BAFK was 6.35 mg/kg. The lipid content from the organisms found in the four negative control replicates on Day 28 of the uptake phase was determined to be 2.17%. The percent organic carbon in the sediment was 1.5%. The normalized Day 28 BAF, based on the lipid content and percent organic carbon in the sediment was 4.42 (OECD 315).

Key value for chemical safety assessment

BMF in fish (dimensionless):

0.003

Additional information

Dietary exposure of fish

GUIDELINE

The objective of this study was to obtain laboratory data characterizing the bioaccumulation potential of test item in the bluegill, Lepomis macrochirus. The protocol was based on procedures outlined in OECD Guidelines for Testing of Chemicals, Guideline 305:Bioaccumulation in Fish: Aqueous and Dietary Exposure.As the BMF is a comparison of the concentration of a substance in an organism with that in the organism’s food, lipid is taken into account by correcting for the contents of lipid in the organism and in the food.

METHODS

The test was divided into two phases: uptake and depuration. During the uptake phase, bluegill were exposed an isotopic mixture of test item in diet at a sub-lethal concentration. The bluegill in the control group were exposed to an untreated diet. The nominal concentration of 500 μg/g test item was selected in consultation with the Sponsor. Each group consisted of one test chamber with 70 fish in each chamber. During the depuration phase, fish were exposed to an untreated diet only. The duration of the uptake phase was 14 days and the depuration phase was 12 days. During both phases of the test, test organisms and water samples were collected and analyzed for 14C radioactivity. These values were used to determine the growth-corrected substance-specific half-life (t1/2g, from the growth-corrected elimination rate constant,k2g), the assimilation efficiency (absorption across the gut; α), the kinetic biomagnification factor (BMFK) and the lipid-corrected kinetic biomagnification factor (BMFKL) for whole fish tissues.

RESULTS

Bluegill were exposed to a control and treatment feed for 14 days. Mean measured concentration of the treatment diet was 497 μg/g test item. The growth and lipid corrected BMF value was 0.0032 in analytically determined whole fish tissues in the treatment group. The measured time zero concentration (tissue concentration at Day 14 uptake) in whole fish tissue was 3.10 μg/g, while the derived time zero concentration in whole fish tissue was 0.268 μg/g suggesting the presence of undigested food in the gut tract. When whole fish tissues were analyzed without the gut tract, the mean measured concentration in fish tissue was 1.15 μg/g.

Exposure of sediment organisms

GUIDELINE

The objective of this study was to determine the bioaccumulation potential of the test item in the oligochaete, Lumbriculus variegatus, through sediment exposure. The protocol was based upon the OECD Guidelines for Testing of Chemicals, Guideline 315: Bioaccumulation in Sediment-dwelling Benthic Oligochaetes

 

METHODS

An initial trial was conducted but was terminated early and repeated due to high mortality in the treatment group. The test concentration was lowered for the definitive test.

 

The test was divided into two phases: the uptake (exposure) phase and the elimination (post-exposure) phase. During the uptake phase, oligochaetes were exposed to one sub-lethal test concentration and a negative and solvent control. The nominal test concentration selected in consultation with the sponsor was 100 mg the test item mg/kg of sediment based on the dry weight of the sediment. Oligochaetes in the solvent control group were exposed under identical conditions without test substance, but with the same amount of solvent used in the treatment group. Oligochaetes in the negative control group were exposed without test substance or solvent. Each test chamber contained the same quantities of sediment and overlying water. Thirty-six replicate test chambers were prepared for both the treatment group and the solvent control group for exposure of the test organisms (i.e. 18 replicates for sampling during the uptake phase plus 18 replicates for sampling during the elimination phase). An additional three replicates were prepared without organisms for the treatment group and the solvent control group for analytical sampling on Day 0. Four replicate test chambers were prepared for the negative control group for sampling at the end of the uptake phase.

 

The results of the study are based on the mean measured test concentrations in the sediment during the uptake phase. The duration of the uptake phase typically will vary according to the time required to reach steady-state, but was not to exceed 28 days. Sediment and overlying water samples were collected and analyzed from three sacrificed replicates of the treatment group and solvent control group on Days 0, 1, 3, 7, 14, 21 and 28 of the uptake phase. Results of the analyses in the sediment were used to verify the exposure over time. Additionally, sediment and overlying water samples were collected on Days 0, 1, 3, 5, 7 and 10 of the elimination phase. The duration of the elimination phase typically will vary according to the time required to reach 10% of the concentration measured in tissues at the end of the uptake phase, but was not to exceed 10 days. Worm tissue samples were collected from the culture on Day 0 and from three replicates from the treatment group and solvent control group on days 1, 3, 7, 14, 21 and 28 of the uptake phase and on days 0, 1, 3, 5, 7 and 10 of the elimination phase. An additional four replicates from the negative control group were sacrificed at the end of the uptake phase for the determination of lipid content in the tissue. The tissue concentrations were used to calculate the uptake rate constant (ks), the elimination rate constant (ke), and the kinetic bioaccumulation factor (BAFK). The bioaccumulation factor (BAF) was calculated, based on the concentration of the test item in the oligochaetes compared to the concentration of the test item in the sediment. Additionally, the residue level in the oligochaetes at the end of the elimination phase (non-eliminated residue; NER) was also determined.

 

RESULTS

The Day 28 bioaccumulation factor (BAF) for oligochaetes (Lumbriculus variegatus) exposed to sediment spiked with the test item at a nominal concentration of 100 mg test substance/kg dry sediment (mean measured concentration during the uptake phase of 92.1 mg the test item equivalents/kg dry sediment) was 6.40. The accumulation in tissue was swift and the elimination was quick but variable. The non-eliminated residue content on Day 10 of the elimination phase was 34.9%. The uptake rate was 0.670 and the elimination rate was 0.105. The BAFK was 6.35 mg/kg. The lipid content from the organisms found in the four negative control replicates on Day 28 of the uptake phase was determined to be 2.17%. The percent organic carbon in the sediment was 1.5%. The normalized Day 28 BAF, based on the lipid content and percent organic carbon in the sediment was 4.42.