Zinc bis(diethyldithiocarbamate)

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Apr 2018 to 16 May 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Remarks:

Preliminary trial

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

October 2012

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1730 (Fish Bioconcentration Test)

Version / remarks:

2016

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): ZDEC
- Zinc content: 18.1%
- Lot Number: 50722504
- Supplier: Chemspec, Ltd.
- Appearance: Solid
- Date Received: September 26, 2017
- Expiration Date: Not provided
- Storage Conditions: Ambient

RADIOLABELLING INFORMATION
- Identity: ZDEC-[14C]
- Radiochemical Purity: 98.28%
- Specific Activity: 140.2 µCi/mg
- Lot Number: 86522-1-21-1
- Supplier: EAG Laboratories - Columbia
- Appearance: Solid
- Date Received: January 9, 2018
- Storage Conditions: Frozen

Radiolabelling:

yes

Details on sampling:

MEDIUM SAMPLING
Water samples were collected and analyzed for total radioactivity from the solvent control and nominal 3.2 µg/L treatment group, 1 and 2 days prior to the start of the exposure period to confirm the concentrations after conditioning the diluter system. In the solvent control and the treatment group, water samples were also collected and analyzed for total radioactivity on days 0 (hours 0 and 4), 1, 5, 7, and 9 of the uptake phase and days 1, 5, 7, and 9 of the depuration phase. On each sampling day, typically one water sample was analyzed from the control test chamber and two from the treatment group test chamber. All water samples for the analysis of total radioactivity were collected in 20-mL glass LSC vials from mid-depth in the test chamber using a glass volumetric pipette. Water samples were processed immediately for analysis. In addition, on Day 12 of the uptake phase, two 1 L water samples from the solvent control and two 1 L water samples from the 3.2 µg/L treatment group were collected. These water samples were then stored frozen for possible analysis of metabolites.

BIOLOGICAL SAMPLING
- Tissue sampling: Tissue samples were collected from the solvent control and the treatment group on uptake days 0 (hour 4), 1, 5, 7, and 9 and on depuration days 1, 5, 7, and 9. At each tissue sampling interval, a sufficient number of fish were collected to provide at least four replicate samples of solvent control fish and four replicate samples 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. Whole fish tissue samples were transferred to pre-weighed containers and weighed. All tissue samples were processed immediately or stored frozen.
- Lipid sampling: Eighteen additional fish were collected from the control and the treatment group to determine lipid content (nine from the control and nine from the treatment group). Fish for lipid analysis were sampled on Day 0 of uptake, at the end of uptake (Day 12 Uptake), and at the end of depuration (Day 9 Depuration). All fish collected for lipid content were stored frozen until analysis.

Vehicle:

yes

Remarks:

0.02 mL/L Dimethylformamide (DMF)

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

Isotopic dilutions were prepared for the dosing stocks. Through the course of the study, four [14C]-test substance primary stock solutions were prepared at a targeted nominal concentration of 1000 mg/L in dimethylformamide (DMF). The first three [14C]-test substance primary stock solutions used in this study were prepared by weighing a calculated amount of radiolabeled test substance into a tared glass beaker, transferring the contents quantitatively into a volumetric flask using HPLC-grade DMF and a glass Pasteur pipette, bringing the flask to volume with DMF, and mixing the solution by inversion. As there was only a small amount of radiolabeled test substance remaining in the test substance container, the last [14C]-test substance primary stock solution was prepared by adding a calculated amount of DMF directly in the test substance container using volumetric pipette and mixed by vortexing. The resulting stock solution was analyzed and diluted again with a calculated volume DMF to bring the stock solution down closer to the targeted nominal concentration of 1000 mg/L. All [14C] primary stock solutions appeared a clear and colorless with no visible signs of precipitate. The radioactivity of each [14C]-test substance primary stock was confirmed prior to use in the study by analyzing three aliquots of the stock solution by liquid scintillation counting (LSC).

Three non-radiolabeled primary stock solutions were prepared during the study by mixing a calculated amount of test substance into HPLC-grade DMF at a nominal concentration of 1.0 mg/mL. The non-radiolabeled primary stock solutions concentrations were not adjusted for the active ingredient of the test substance during preparation, and all concentration are based on the test substance as received. The non-radiolabeled primary stock solutions were sonicated for approximately 15 minutes followed by mixing by inversion. The non-radiolabeled primary stock solutions appeared clear and orange, with no visible precipitates.

The dispensing stocks were prepared daily at a nominal concentration of 0.16 mg/mL. The dispensing stocks were prepared by mixing the appropriate volumes of radiolabeled and non-radiolabeled test substance primary stock solutions and bringing the stocks to volume with DMF. The radioactivity of the dispensing stock solutions was approximately 30,000,000 dpm/mL. The final nominal radioactivity in the treatment group aqueous exposure solution was 600 dpm/mL. Dispensing stocks were prepared in 12 mL volumes, mixed by inversion and appeared clear and colorless. The final specific activity in the treatment group ranged from 187,046 to 187,536 dpm/µg.

Each dispensing stock solution was placed in a syringe daily during the uptake phase of the study. The dispensing stocks were pumped into the diluter mixing chamber assigned to the treatment group at a target rate of 7.00 µL/min and were mixed with well water in the mixing chamber, delivered at a target rate of 350 mL/min, to achieve the nominal test concentration of 3.2 µg/L. The solvent control was prepared by delivering DMF to the mixing chamber for the solvent control. The concentration of DMF in the control and treatment groups was 0.02 mL/L. Dilution water was delivered at a target rate of 197 mL/min during the depuration phase. Flows to the test system were initiated nine days prior to initiation of the uptake phase. The test solutions in the mixing chambers and test chambers appeared clear and colorless during both the uptake and depuration phases.

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name: Bluegill
- Source: Osage Catfisheries, Inc., Osage Beach, Missouri
- Age: Juveniles (hatched on April 18, 2017)
- Length at study initiation: 5.5 cm (5.0 - 5.9 cm; std. dev = 0.3 cm)
- Weight at study initiation (mean and range, SD): 2.28 g (1.72 - 2.77 g; std. dev = 0.328 g)
- Feeding: Fed at least once daily during holding with a commercially-prepared diet supplied by Sera North America, Inc.
- Mortality prior to test: None

ACCLIMATION
- Acclimation period: The fish were held for at least 14 days prior to the test.
- Acclimation conditions: Fish were held in water from the same source and at approximately the same temperature as used during the test. During the 2 week period immediately preceding the test, water temperatures ranged from 20.0 to 23.5 °C, the pH of the water ranged from 8.3 to 8.4 and the dissolved oxygen remained ≥8.2 mg/L (≥94% of saturation).
- Health during acclimation: All fish in the culture during this time appeared normal, with no mortalities during the 14-day holding period.
- Feeding: During the holding period and the test, fish were fed at least once daily a commercially prepared diet of Sera Vipan supplied by Sera North America of Montgomeryville, Pennsylvania. Uneaten food and feces were siphoned daily from the test chambers shortly after feeding. The diet was fed at a rate of 2% of fish body weight. To ensure that the feeding rate per fish remained constant, the amount of food supplied to each test chamber was adjusted at least weekly based on weight of the fish. Specifications for acceptable levels of contaminants in fish diets have not been established. However, there were no known levels of contaminants reasonably expected to be present in the diet that were considered to interfere with the purpose or conduct of the test.

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason: See 'Details on test conditions' (range-finding / preliminary study)

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

12 d

Total depuration duration:

9 d

Hardness:

128 - 136 mg/L as CaCO3

Test temperature:

Continuous measurements of temperature (measured with an Fulscope ER/C Recorder to the nearest 0.5 °C) ranged from 21 to 23 °C during the uptake phase and from 21 to 22 °C during the depuration phase.

pH:

8.0 - 8.6

Dissolved oxygen:

7.0 - 8.7 mg O2/L

TOC:

UPTAKE PHASE
- Dilution water: <1 - 1.1 mg C/L
- Solvent control: 8.44 - 9.08 mg C/L
- Treatment: 6.71 - 8.96 mg C/L

DEPURATION PHASE
- Dilution water: <1 - 1.412 mg C/L
- Solvent control: <1 - <1 mg C/L
- Treatment: <1 - 2.178 mg C/L

Conductivity:

343 - 385 µS/cm

Details on test conditions:

TEST SYSTEM
- Test vessel uptake phase: Test chambers were positioned in temperature-controlled water baths designed to maintain the target temperature throughout the test period. Test chambers used during the uptake phase were 127-L Teflon-lined stainless steel aquaria filled with 80 L of test solution. The depth of the test water in a representative test chamber during uptake was approximately 18.5 cm.
- Test vessel depuration phase: On Day 12 of the uptake phase, the fish were transferred to a clean set of 54-L Teflon-lined stainless steel aquaria filled with 45 L of dilution water (well water) for the depuration phase. The depth of the test water in a representative test chamber during depuration was approximately 26.4 cm.
- Type: Open. Test chambers were siphoned daily during the test to remove excess feed, fecal matter, algae and bacterial growth.
- Type of flow-through: The bioconcentration test was conducted using an exposure system consisting of continuous flow diluters to deliver each concentration of the test substance and a solvent (dimethylformamide (DMF)) control to test chambers. Syringe pumps (Harvard Apparatus, Holliston, Massachusetts) were used to deliver volumes of test substance stock solutions and the solvent control to mixing chambers impartially assigned to the treatment and control groups. The stock solutions were diluted with well water in the mixing chambers in order to obtain the desired test concentrations prior to delivery to the test chambers. The flow of dilution water into each mixing chamber was controlled using rotameters, and was adjusted to provide approximately six volume additions of test solution in each test chamber per day. The general operation of the diluter was checked at least two times a day during the test, and at least once at the beginning and end of each phase of the test. The pumps used to deliver stock solutions and solvent to the mixing chambers were calibrated prior to the test. The rotameters used to control the flow of dilution water to the mixing chambers were calibrated prior to the test and verified weekly throughout the uptake and depuration phases.
- No. of organisms per vessel: 100
- No. of vessels per concentration: 1; two separate water baths of the same type were used for the uptake and depuration phases.
- No. of vessels per vehicle control: 1 ; two separate water baths of the same type were used for the uptake and depuration phases.
- Biomass loading rate: Biomass loading rates were calculated using the wet weights of ten fish collected on Day 0 since this was considered to be representative of the biomass in test chambers during the test. All fish used in the test were from the same source and year class. Loading was defined as the total wet weight of fish per liter of test water that passed through the test chamber in 24 hours, and was determined to be 0.45 g fish/L/day. Instantaneous loading (the total wet weight of fish per liter of water in the tank) was 2.9 g fish/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 EAG Laboratories-Easton site. The well water is characterized as moderately hard water. The well water was passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800-L storage tank and aerated with spray nozzles. Prior to use, the water was filtered to 0.45 µm to remove fine particles and was passed through an ultraviolet (UV) sterilizer.
- Alkalinity: 168 - 182 mg/L as CaCO3
- Holding medium different from test medium: Same

OTHER TEST CONDITIONS
- Adjustment of pH: No
- 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 quality: Fluorescent light bulbs that emit wavelengths similar to natural sunlight were used for illumination of the test chambers.
- Light intensity: Light intensity at the surface of the water at the start of the uptake phase was 785 lux. Light intensity at the surface of the water at the start of the depuration phase was 928 lux. Light intensity was measured using a SPER Scientific Model 840006 light meter.

WATER QUALITY MEASUREMENTS
- Temperature: Temperature was measured in the test chambers at the beginning and end of the uptake and depuration phases and at approximately weekly intervals during the test using a digital thermometer. Temperature was also monitored continuously in the solvent control test chamber using a Fulscope ER/C Recorder, which were verified prior to test initiation and at approximately weekly intervals thereafter using a digital thermometer.
- Dissolved oxygen: Dissolved oxygen measurements were made in each test chamber daily during the uptake and depuration phases using a Thermo Scientific Orion Star A213 dissolved oxygen 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: Hardness, alkalinity and conductivity were measured in the dilution water, the solvent control, and in the treatment group at the beginning and end of the uptake and depuration phases. Total organic carbon (TOC) was measured in the dilution water, the solvent control, and in the treatment group at the beginning of the uptake and depuration phases prior to the addition of the fish, at the end of the uptake and depuration phases, and approximately weekly during the uptake and depuration phases. 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. Total organic carbon was measured using a Shimadzu model TOC-VCSH total organic carbon analyzer.

BIOLOGICAL MEASUREMENTS
Daily observations of mortality, unusual behavior, and overall appearance were made during the uptake and depuration phases. Any mortalities or external abnormalities (such as hemorrhaging and discoloration) were noted.

RANGE-FINDING / PRELIMINARY STUDY
A preliminary trial was conducted at a nominal concentration of 3.2 µg/L to estimate the uptake and depuration for ZDEC (See the supporting study in IUCLID Section 5.3.1). The test concentration was based on the No Observed Effect Value of 3.2 µg/L for an 11-day toxicity test conducted on Danio rerio, which is approximately 1% of the 96-hour LC50 values calculated for Oncorhynchus mykiss (0.23 mg/L) and Poecilia reticulata (0.49 mg/L). Based on the non-GLP trial, the time to steady state was estimated to be approximately 18 days and the time to reach 50% clearance estimated to be approximately 4.3 days. The estimated kinetic BCF (BCFK) value in whole fish tissue using the data from the preliminary trial was 10. Based on the low uptake of the test substance in whole fish tissues in the pilot study it was determined that measuring the test substance concentrations in edible and non-edible tissues would be problematic. As such, it was determined that only whole fish tissues were to be analyzed in the definitive study. Moreover, it was determined that the addition of a second test concentration to the definitive BCF study would be infeasible as the high test solution activity that would be required to reliably measure the test substance throughout the study and the specific activity of the radiolabeled test substance meant that the test substance concentration could be not lowered below approximately 2.0 µg/L, which is minimally different from the nominal test concentration of 3.2 µg/L. Therefore, a single nominal test concentration of 3.2 µg/L chosen for the definitive test.

Nominal and measured concentrations:

- Nominal concentrations: 0 (solvent control) and 3.2 µg/L
- Mean measured concentrations:

Reference substance (positive control):

no

Details on estimation of bioconcentration:

BASIS INFORMATION
- Measured/calculated logPow: 3.11

Lipid content:

5.11 %

Time point:

start of exposure

Remarks on result:

other: Solvent control

Remarks:

Results are tabulated in 'Any other information on results in tables'

Lipid content:

6.91 %

Time point:

end of exposure

Remarks on result:

other: Solvent control

Remarks:

Results are tabulated in 'Any other information on results in tables'

Key result

Conc. / dose:

3.2 µg/L

Temp.:

>= 21.7 - <= 22.1 °C

pH:

8.3

Type:

BCF

Value:

20 L/kg

Basis:

other: whole fish tissue

Time of plateau:

12 d

Calculation basis:

kinetic

Remarks on result:

other: BCF(K)

Conc. / dose:

3.2 µg/L

Temp.:

>= 21.7 - <= 22.1 °C

pH:

8.3

Type:

BCF

Value:

21 L/kg

Basis:

other: whole fish tissue

Time of plateau:

12 d

Calculation basis:

kinetic, corrected for growth

Remarks on result:

other: BCF(Kg)

Conc. / dose:

3.2 µg/L

Temp.:

>= 21.7 - <= 22.1 °C

pH:

8.3

Type:

BCF

Value:

13 L/kg

Basis:

other: whole fish tissue

Time of plateau:

12 d

Calculation basis:

steady state

Remarks on result:

other: BCF(SSL)

Conc. / dose:

3.2 µg/L

Temp.:

>= 21.7 - <= 22.1 °C

pH:

8.3

Type:

BCF

Value:

15 L/kg

Basis:

normalised lipid fraction

Time of plateau:

12 d

Calculation basis:

kinetic

Remarks on result:

other: BCF(KL)

Conc. / dose:

3.2 µg/L

Temp.:

>= 21.7 - <= 22.1 °C

pH:

8.3

Type:

BCF

Value:

15 L/kg

Basis:

normalised lipid fraction

Time of plateau:

12 d

Calculation basis:

kinetic, corrected for growth

Remarks on result:

other: BCF(KgL)

Elimination:

yes

Parameter:

DT50

Remarks:

growth-rate corrected (t1/2g)

Depuration time (DT):

5.2 d

Rate constant:

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

Remarks:

k1(days)

Value:

2.79

Rate constant:

overall depuration rate constant (d-1)

Remarks:

k2 (days)

Value:

0.139

Rate constant:

growth rate constant (d-1)

Remarks:

kg

Value:

0.005

Rate constant:

growth-corrected depuration rate constant (d-1)

Remarks:

k2g

Value:

0.134

Rate constant:

growth-corrected half-life (d)

Remarks:

t1/2g (days)

Value:

5.2

Metabolites:

Not reported

Details on results:

MORTALITY
No mortalities or abnormal behavior were observed in the solvent control group. There was one mortality in treatment group on Day 6 of the uptake phase, but all other fish appeared normal throughout the study. The sole mortality in the treatment group was considered incidental. At the end of the uptake phase (Day 12), the fish were enumerated during the transfer to depuration tanks.

CONCENTRATIONS OF TOTAL RADIOACTIVITY IN WATER
The results on the analytical verifications are tabulated in 'Any other information on results incl.tables'.
The measured concentrations of total radioactivity in the pre-test samples collected 2 days prior to initiation ranged from approximately 101 to 102% of nominal concentrations. Aeration was added to the test system after collection of the Day 2 samples. The measured concentrations of total radioactivity in the pre-test samples collected 1 day prior to initiation ranged from approximately 98.5 to 100% of nominal. The measured concentrations of total radioactivity ranged from approximately 98.7 to 103% of the treatment group’s nominal concentration, with an overall mean measured percent of nominal of 100%. The results of the study were based on the time-weighted mean measured test concentrations of 3.2 µg/L for the uptake phase. The measured concentrations of total radioactivity in water during the depuration phase were all below the limit of quantitation.

CONCENTRATIONS OF TOTAL RADIOACTIVITY IN FISH
The concentrations of total radioactivity in tissues of fish exposed to time-weighted mean measured test concentrations of 3.2 µg/L (nominal concentration of 3.2 µg/L) are presented in 'Any other information on results incl.tables'. The mean measured steady-state whole fish tissue concentration based on total radioactivity was 39.3 µg/kg.

STEADY STATE
It was determined that steady state was reached on days 5, 7, and 9 in in the treatment group. The steady-state BCF value for the treatment group, based on total radioactivity, was 13 in whole fish tissue. The estimated time to reach 95% of steady state (t95) in whole fish was 22 days. The estimated time to reach 50% clearance (t1/2 ) in whole fish tissue was 5 days.

Reported statistics:

The steady-state bioconcentration factor (BCF) values were determined from the mean tissue concentrations (based on total radioactivity) at the last interval of apparent steady-state divided by the time-weighted mean measured water concentration. Tissue concentrations were considered to be at apparent steady-state if three or more consecutive sets of tissue concentrations are within ±20% of each other, and there is no significantly increasing trend in residue concentration in time between the first and last successive analyses (p > 0.05) as determined using the Jonckheere-Terpstra trend test. All statistical tests were performed using a personal computer with The SAS System for Windows software. Nonlinear regression was used to sequentially solve for k1 and k2 using fish tissue data from the uptake phase and depuration phase. These rate constants were used to calculate a kinetic bioconcentration factor (BCFK = k1/k2) and also to calculate half-life for clearance in tissue (t½) and, time to reach 95% of steady state (t95).

The kinetic uptake and depuration rate constants were determined using nonlinear regression described by Newman. In the sequential method, data from the depuration (elimination) phase was used to first estimate k2, and then using both the k2 estimate and fish tissue from the uptake phase to estimate k1. In addition, lipid-normalized and growth-corrected kinetic and steady state bioconcentration factors (BCFSSL, BCFKL, BCFKg and BCFKgL) as well as growth corrected half-life (t½g) were determined.

Table: Measured Concentrations of Total Radioactivity in Water Samples During the Uptake Phase (Measured by LSC)

Nominal Test substance concentration (µg/L)	Sampling interval	Specific Activity (dpm/µg)	Total [14C] found (dpm)	Test substance Equivalents found (1,2,3,4) (µg/L)	Percent of nominal (4)
--	0	--	0.00	--	--
--	1	--	0.00	--	--
--	5	--	0.00	--	--
--	7	--	0.00	--	--
--	9	--	0.00	--	--
0.0	0 (Hour 0)	187536	33.11	< LOQ	--
0.0	0 (Hour 4)	187536	24.00	< LOQ	--
0.0	1	187536	5.68	< LOQ	--
0.0	5	187046	11.98	< LOQ	--
0.0	7	187413	18.00	< LOQ	--
0.0	9	187413	24.11	< LOQ	--
3.2	0 (Hour 0)	187536	6131.16	3.27	102
3.2	0 (Hour 0)	187536	5995.06	3.20	99.9
3.2	0 (Hour 4)	187536	5920.50	3.16	98.7
3.2	0 (Hour 4)	187536	5959.68	3.18	99.3
3.2	1	187536	6022.83	3.2	100
3.2	1	187536	5996.72	3.2	99.9
3.2	5	187046	6031.83	3.22	101
3.2	5	187046	6164.99	3.30	103
3.2	7	187413	5988.29	3.20	99.9
3.2	7	187413	5994.22	3.20	100
3.2	9	187413	5995.52	3.20	100
3.2	9	187413	5916.58	3.16	98.7
Mean (4):				3.2 ± 0.03 (CV 1.1%)	100%
Time‑weighted Mean (4):				3.2 ± 0.01 (CV = 0.37%)	101%

1) The sample volume was 10 mL (10 mL for background samples).

2) Test substance Equiv. = {[(Total dpm found)/Sample Volume]/Specific Activity} x 1000 mL/L.

3) The limit of quantitation (LOQ) for freshwater samples was based on the instrument LOQ of 50 dpm. Any sample with a total dpm count below 50 was labeled as <LOQ.

4) Results generated using Excel 2010. Manual calculations may differ slightly.

Note: The background contribution was automatically subtracted by the software for the LSC.

 

Table: Measured Concentrations of Total Radioactivity in Water Samples During the Depuration Phase (Measured by LSC)

Nominal Test substance Concentration (µg/L)	Sampling Interval (Day)	Specific Activity (dpm/µg)	Total [14C] Found (dpm)	Test substance Equivalents Found (1,2,3,4) (µg/L)	Percent of Nominal (4)
--	1	--	0.00	--	--
--	5	--	0.00	--	--
--	7	--	0.00	--	--
--	9	--	0.00	--	--
0.0	1	187529	14.43	< LOQ	--
0.0	5	187529	15.80	< LOQ	--
0.0	7	187529	23.17	< LOQ	--
0.0	9	187529	3.27	< LOQ	--
3.2	1	187529	22.36	< LOQ	--
3.2	1	187529	26.54	< LOQ	--
3.2	5	187529	26.02	< LOQ	--
3.2	5	187529	30.85	< LOQ	--
3.2	7	187529	19.37	< LOQ	--
3.2	7	187529	31.13	< LOQ	--
3.2	9	187529	16.94	< LOQ	--
3.2	9	187529	10.87	< LOQ	--

1) The sample volume was 10 mL (10 mL for background samples).

2) Test substance Equiv. = {[(Total dpm found)/Sample Volume]/Specific Activity} x 1000 mL/L.

3) The limit of quantitation (LOQ) for freshwater samples was based on the instrument LOQ of 50 dpm. Any sample with a total dpm count below 50 was labeled as <LOQ.

4) Calculated using Excel 2010 in full precision mode. Manual calculations may differ slightly.

Note: The background contribution was automatically subtracted by the software for the LSC.

 

Table: Time-Weighted Mean Measured Concentrations of Total Radioactivity in Water Samples by Day During the Uptake and Depuration Phases (Measured by LSC)

 

Nominal Test subtance Concentration (µg/L)	Study Phase	Sampling Interval (Day)	Test substance Mean Equivalents Found (1,2,3,4) (µg/L)	Number of Samples	Percent of Nominal(4)	Time-Weighted Mean Measured Concentration (µg/L)(4)
0	Uptake	0 (Hour 0)	<LOQ	1	--	--
0	Uptake	0 (Hour 4)	<LOQ	1	--
0	Uptake	1	<LOQ	1	--
0	Uptake	5	<LOQ	1	--
0	Uptake	7	<LOQ	1	--
0	Uptake	9	<LOQ	1	--
3.2	Uptake	0 (Hour 0)	3.24	2	101	3.2
3.2	Uptake	0 (Hour 4)	3.17	2	99.1
3.2	Uptake	1	3.21	2	100
3.2	Uptake	5	3.26	2	102
3.2	Uptake	7	3.20	2	100
3.2	Uptake	9	3.18	2	99.4
0	Depuration	1	<LOQ	1	--	--
0	Depuration	5	<LOQ	1	--
0	Depuration	7	<LOQ	1	--
0	Depuration	9	<LOQ	1	--
3.2	Depuration	1	<LOQ	2	--	--
3.2	Depuration	5	<LOQ	2	--
3.2	Depuration	7	<LOQ	2	--
3.2	Depuration	9	<LOQ	2	--

1) The sample volume was 10 mL (10 mL for background samples).

2) Test substance Equiv. = {[(Total dpm found)/Sample Volume]/Specific Activity} x 1000 mL/L.

3) The limit of quantitation (LOQ) for freshwater samples was based on the instrument LOQ of 50 dpm. Samples with a total dpm count below 50 were labeled as <LOQ.

4) Results generated using Excel 2010 in full precision mode. Manual calculations may differ slightly.

 

Table: Measured Concentrations of Total Radioactivity in Whole Fish Tissues

During the Uptake Phase (Measured by LSC)

 

Sample Type	Sampling Interval (Day)	Nominal Test substance Concentration (µg/L)	Sample Mass (g)	Specific Activity (dpm/µg)	Total [14C] Found (dpm)	Test substance Equivalents Found (µg/kg) (1,2)
Background	--	--	NA	--	0.00	--
Whole Fish	0	0	0.350	187536	0.00	< LOQ
Whole Fish	0	0	0.350	187536	0.00	< LOQ
Whole Fish	0	0	0.350	187536	0.00	< LOQ
Whole Fish	0	0	0.350	187536	0.00	< LOQ
Whole Fish	0	3.2	0.350	187536	358.68	5.46
Whole Fish	0	3.2	0.350	187536	524.58	7.99
Whole Fish	0	3.2	0.350	187536	680.77	10.4
Whole Fish	0	3.2	0.350	187536	379.73	5.79
Background	--	--	NA	--	0.00	--
Whole Fish	1	0	0.350	187536	0.00	< LOQ
Whole Fish	1	0	0.350	187536	0.00	< LOQ
Whole Fish	1	0	0.350	187536	0.81	< LOQ
Whole Fish	1	0	0.350	187536	0.00	< LOQ
Whole Fish	1	3.2	0.350	187536	1138.04	17.3
Whole Fish	1	3.2	0.350	187536	1144.63	17.4
Whole Fish	1	3.2	0.350	187536	782.47	11.9
Whole Fish	1	3.2	0.350	187536	1276.88	19.5
Background	--	--	NA	--	0.00	--
Whole Fish*	5	0	0.350	187046	13.62	<LOQ
Whole Fish*	5	0	0.350	187046	16.85	<LOQ
Whole Fish	5	0	0.350	187046	16.02	< LOQ
Whole Fish	5	0	0.350	187046	0.22	< LOQ
Whole Fish	5	3.2	0.350	187046	2568.09	39.2
Whole Fish	5	3.2	0.350	187046	2258.13	34.5
Whole Fish	5	3.2	0.350	187046	2731.95	41.7
Whole Fish	5	3.2	0.350	187046	2148.83	32.8
Background	--	--	NA	--	0.00	--
Whole Fish	7	0	0.350	187413	10.57	< LOQ
Whole Fish	7	0	0.350	187413	11.54	< LOQ
Whole Fish	7	0	0.350	187413	4.19	< LOQ
Whole Fish	7	0	0.350	187413	8.31	< LOQ
Whole Fish	7	3.2	0.350	187413	3354.62	51.1
Whole Fish	7	3.2	0.350	187413	2139.93	32.6
Whole Fish	7	3.2	0.350	187413	2377.69	36.2
Whole Fish	7	3.2	0.350	187413	2550.99	38.9
Background	--	--	--		0.00	--
Whole Fish	9	0	0.350	187413	10.83	< LOQ
Whole Fish	9	0	0.350	187413	0.00	< LOQ
Whole Fish	9	0	0.350	187413	0.00	< LOQ
Whole Fish	9	0	0.350	187413	1.48	< LOQ
Whole Fish	9	3.2	0.350	187413	2292.38	34.9
Whole Fish	9	3.2	0.350	187413	2411.37	36.8
Whole Fish	9	3.2	0.350	187413	2842.15	43.3
Whole Fish	9	3.2	0.350	187413	3257.30	49.7

1) Equivalents found = ((Total dpm found)/Sample Mass))/Specific Activity x 1000 g/kg

2) The limit of quantitation (LOQ) for tissue samples was based on the instrument LOQ of 50 dpm. Any sample with a total dpm count below 50 was labeled as <LOQ.

*The reanalysis results for these samples are presented.

Note: The background contribution was automatically subtracted by the software for the LSC.

 

Table: Measured Concentrations of Total Radioactivity in Whole Fish Tissues

During the Depuration Phase (Measured by LSC)

Sample Type	Sampling Interval (Day)	Nominal Test substance Concentration (µg/L)	Sample Mass (g)	Specific Activity (dpm/µg)	Total [14C] Found (dpm)	Test substance Equivalents Found (µg/kg) (1,2)
Background	--	--	NA	--	0.00	--
Whole Fish	1 Dep.	0	0.350	187529	10.31	< LOQ
Whole Fish	1 Dep.	0	0.350	187529	3.16	< LOQ
Whole Fish	1 Dep.	0	0.350	187529	0.83	< LOQ
Whole Fish	1 Dep.	0	0.350	187529	8.59	< LOQ
Whole Fish	1 Dep.	3.2	0.350	187529	2422.35	36.9
Whole Fish	1 Dep.	3.2	0.350	187529	1762.95	26.9
Whole Fish	1 Dep.	3.2	0.350	187529	1337.91	20.4
Whole Fish	1 Dep.	3.2	0.350	187529	963.61	14.7
Background	--	--	NA	--	0.00	--
Whole Fish	5 Dep.	0	0.350	187529	0.00	< LOQ
Whole Fish	5 Dep.	0	0.350	187529	0.00	< LOQ
Whole Fish	5 Dep.	0	0.350	187529	0.00	< LOQ
Whole Fish	5 Dep.	0	0.350	187529	0.00	< LOQ
Whole Fish	5 Dep.	0	0.350	187529	0.00	< LOQ
Whole Fish	5 Dep.	3.2	0.350	187529	784.60	12.0
Whole Fish	5 Dep.	3.2	0.350	187529	1208.26	18.4
Whole Fish	5 Dep.	3.2	0.350	187529	1296.02	19.7
Whole Fish	5 Dep.	3.2	0.350	187529	795.79	12.1
Whole Fish	5 Dep.	3.2	0.350	187529	705.14	10.7
Background	--	--	NA	--	0.00	--
Whole Fish	7 Dep.	0	0.350	187529	0.00	< LOQ
Whole Fish	7 Dep.	0	0.350	187529	0.00	< LOQ
Whole Fish	7 Dep.	0	0.350	187529	0.00	< LOQ
Whole Fish	7 Dep.	0	0.350	187529	0.00	< LOQ
Whole Fish	7 Dep.	3.2	0.350	187529	779.88	11.9
Whole Fish	7 Dep.	3.2	0.350	187529	884.27	13.5
Whole Fish	7 Dep.	3.2	0.350	187529	852.07	13.0
Whole Fish	7 Dep.	3.2	0.350	187529	779.44	11.9
Background	--	--	NA	--	0.00	--
Whole Fish	9 Dep.	0	0.350	187529	1.51	< LOQ
Whole Fish	9 Dep.	0	0.350	187529	1.89	< LOQ
Whole Fish	9 Dep.	0	0.350	187529	7.00	< LOQ
Whole Fish	9 Dep.	0	0.350	187529	3.25	< LOQ
Whole Fish	9 Dep.	3.2	0.350	187529	713.46	10.9
Whole Fish	9 Dep.	3.2	0.350	187529	752.10	11.5
Whole Fish	9 Dep.	3.2	0.350	187529	573.49	8.7
Whole Fish	9 Dep.	3.2	0.350	187529	640.04	9.8

1) Equivalents found = ((Total dpm found)/Sample Mass))/Specific Activity x 1000 g/kg

2) The limit of quantitation (LOQ) for tissue samples was based on the instrument LOQ of 50 dpm. Any sample with a total dpm count below 50 was labeled as <LOQ.

Note: The background contribution was automatically subtracted by the software for the LSC.

 

Table: Mean Measured Concentrations of Total Radioactivity in Whole Fish Tissues by Day During the Uptake and Depuration Phases (Measured by LSC)

Nominal Test substance Concentration (µg/L)	Study Phase	Sampling Interval (Day)	Mean Whole Fish Conc. (1,2,3) (µg/kg)
0	Uptake	0 (Hour 4)	<LOQ
0	Uptake	1	<LOQ
0	Uptake	5	<LOQ
0	Uptake	7	<LOQ
0	Uptake	9	<LOQ
3.2	Uptake	0 (Hour 4)	7.41
3.2	Uptake	1	16.5
3.2	Uptake	5	37.1
3.2	Uptake	7	39.7
3.2	Uptake	9	41.2
0	Depuration	1	<LOQ
0	Depuration	5	<LOQ
0	Depuration	7	<LOQ
0	Depuration	9	<LOQ
3.2	Depuration	1	24.7
3.2	Depuration	5	14.6
3.2	Depuration	7	12.6
3.2	Depuration	9	10.2

1) µg/kg concentrations are expressed as equivalents of test substance based on specific activity.

2) Results were generated using rounded tissue concentrations in Excel 2010. Manual calculations may differ slightly.

3) The limit of quantitation (LOQ) for tissue samples was based on the instrument LOQ of 50 dpm. Samples with a total dpm count below 50 were labeled as <LOQ.

 

Table: Confirmation of Steady State

Tissue Type	Nominal Water Concentration (1) (µg/L)	Mean Measured Tissue Concentration (µg/kg)2,3 For the Last Three Sampling Intervals			Mean(3)	SD(3)	p(4)
		Day5	Day 7	Day 9
Whole Fish	3.2	37.1	39.7	41.2	39.3	2.1	0.2321

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

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

3) Calculated using Excel 2010. Manual calculations may differ slightly.

4) α = 0.05; p-value calculated using one-sided Jonckheere-Terpstra trend test in SAS.

 

Table: Steady-State BCF Values for Bluegill Based on Total Radioactivity

Tissue Type	Time-Weighted Mean Measured Test Concentration (µg/L) 1,2	Uptake Days at Steady State	Apparent Steady-State Tissue Concentration 2,3,4 (µg/kg)	Tissue Concentration at Last Interval of Steady-State	Steady-State BCF2,4	BCFSS 95% Confidence Interval2
Whole Fish	3.2	5-7-9	39.3	41.2	13	9 – 16

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

2) Calculated using Excel 2010.

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

4) Steady-State BCF = (mean tissue concentration at last interval of steady-state)/(time-weighted mean measured water concentration). Although apparent steady-state is confirmed over three intervals, only the mean tissue concentration for the last steady-state interval is used for the BCFSS calculation.

 

Table: Kinetic Estimates of the BCFK for Bluegill Based on Total Radioactivity

Tissue Type	Nominal Exposure (µg/L)	Uptake Rate Constant (k1, Day-1) (1)	Depuration Rate Constant (k2, Day-1) (1)	Kinetic Bioconcentration Factor (BCFK) (2)	BCFK 95% Confidence Interval (3)	Estimated Time to Reach 95% Steady State (Days) (2)	Estimated Time to Reach 50% Clearance (Days) (2)
Whole Fish	3.2	2.79	0.139	20	15 - 26	22	5

1) Calculated using SAS.

2) Calculated in Excel 2010 using methods outlined in OPPTS guideline 850.1730.

3) Calculated using Excel 2010 according to methods described by Bailer et al. 2000. Estimated and Testing Bioconcentration Factors. Environmental Toxicology and Chemistry, Vol. 19 No. 9, pg. 2338-2340.

 

Table: Growth rate constants

	kg values
	Solvent Control(1)	3.2 µg/L(1)
Uptake Phase	-0.0303	0.00336
Depuration Phase	0.0349	-0.0113
Pooled Uptake and Depuration Phases^2	0.00386	0.00594

1) No statistical difference between growth rate constants of uptake and depuration phases for the solvent control or the single treatment group (p>0.05).

2) No statistical difference between solvent control and the treatment group for growth rate constants using pooled uptake and depuration phase data.

3) Growth measurements from both the uptake and depuration phases of the control and the single treatment group were pooled for the overall growth rate constant used for growth correction calculations. 

 

Table: Growth-Corrected Values in Whole Fish Tissues

	3.2 µg/L
k2	0.139
kg	0.00490
k2g	0.134
Ln	0.724
BCF(Kg)	21
BCF(KL)	15
BCF(KgL)	15
BCF(SSL)	9
t1/2g	5.2

k2 = depuration rate constant.

kg = growth rate constant.

k2g = growth corrected depuration rate constant.

Ln = Lipid normalization factor.

t1/2g = growth corrected half- life (days).

BCF(SSL) = lipid-normalized steady state BCF value.

BCF(Kg) = growth corrected kinetic BCF value.

BCF(KL) = lipid-normalized kinetic BCF value.

BCF(KgL) = growth and lipid corrected kinetic BCF value.

 

Table: Whole Fish Lipid Determination

Treatment Group (μg/L)	Sampling Interval (Day)	Tissue Weight (grams)	Lipid Weight (grams)	Percent Lipids (%)	Mean Percent Lipids(1)
Solvent Control	Uptake Day 0	2.6562	0.138	5.18	5.11
Solvent Control	Uptake Day 0	2.2904	0.107	4.68
Solvent Control	Uptake Day 0	2.7674	0.151	5.46
Solvent Control	12 Uptake/0 Dep.	2.1965	0.136	6.17	6.91 (Ln2 = 0.724)
Solvent Control	12 Uptake/0 Dep.	2.2956	0.167	7.26
Solvent Control	12 Uptake/0 Dep.	2.2082	0.161	7.30
Solvent Control	Dep. Day 9	2.7398	0.225	8.21	6.76
Solvent Control	Dep. Day 9	2.0747	0.082	3.95
Solvent Control	Dep. Day 9	3.3399	0.271	8.13

1) Results were generated using Excel 2010 in full precision mode. Manual calculations may differ slightly.

2) Ln = lipid normalization factor based on lipid content of the fish at the end of uptake.

Validity criteria fulfilled:

yes

Remarks:

see 'Any other information on materials and methods incl. tables'.

Description of key information

The kinetic BCF value of the test substance is determined to be 20 L/kg in an experimental study according to OECD TG 305-I with bluegill fish (Lepomis macrochirus).

Key value for chemical safety assessment

BCF (aquatic species):

20 L/kg ww

Additional information

The bioaccumulative potential of the test substance in freshwater fish is determined in a GLP-compliant experimental study in accordance with OECD TG 305-I (aqueous exposure) and OPPTS guideline 850.1730 (EAG Inc., 2019). In this study, bluegill fish (Lepomis macrochirus; 100 fish per treatment; maximum loading 0.45 g fish/L/day) were exposed to a solvent control and 3.2 µg/L of radiolabelled test substance under flow-though conditions for 12 days during the uptake phase (steady state) before being transferred to clean test chambers containing only dilution water for another 9 days (depuration phase). Measured concentrations of the test item in water before and during the uptake phase remained will within ± 20% of the nominal concentration. The test item was not detected (<LOQ) in the depuration phase. Daily observations of mortality, unusual behavior, and overall appearance were made during the uptake and depuration phases. Water quality parameters were monitored at the beginning and end of the uptake and depuration phases.

 

No mortalities or abnormal behavior were observed in the solvent control group. There was one mortality in treatment group on Day 6 of the uptake phase, but all other fish appeared normal throughout the study. The mean measured steady-state whole fish tissue concentration based on total radioactivity was 39.3 µg/kg. The mean measured whole fish tissue concentration based on total radioactivity at termination of the depuration phase (Day 9) was 10.2 µg/kg which is approximately 26% of the apparent mean measured steady-state tissue concentration. The data collected from the uptake and depuration phases were used to calculate the uptake rate constant (k1), 2.79 per day; the depuration rate constant (k2), 0.139 per day; the kinetic bioconcentration factor (BCF(K)), 20 L/kg; the time to reach 95% steady state, 22 days; and the time to 50% clearance, 5.2 days. The results are well in line with the preliminary study; which determined an uptake rate constant (k1) of 1.62 per day; a depuration rate constant (k2) of 0.163 per day; a kinetic bioconcentration factor (BCF(K)) of 9.97 L/kg with a time to reach 95% steady state, of18.4 days and the time to reach 50% clearance of 4.3 days.

 

Note on deviation/reliability

Analyses from whole fish tissues collected on Day 7 of the depuration phase showed that concentrations were <LOQ. As such, the in-life phase was terminated on Day 9 of the depuration phase. During the data quality check that occurred after termination, an error in the transcription of the total dpm counts for whole fish tissues from Day 7 of the depuration phase was discovered. It was determined that ZDEC concentrations in whole fish tissues were >LOQ and that the mean measured whole fish tissue concentration, based on total radioactivity, was actually 12.6 µg/kg, which is approximately 39.3% of the apparent mean measured steady-state tissue concentration. Subsequent analysis of backup tissue samples collected at the termination of the in-life phase (Day 9 Depuration) showed that the mean whole fish tissue concentration, based on total radioactivity, had dropped to 10.2 µg/kg which is approximately 26% of the apparent mean measured steady-state tissue concentration. Therefore, the depuration phase was inadvertently terminated prematurely on Day 9 of the depuration phase. Although the test was terminated before ZDEC concentrations in whole fish tissues dropped below 5% of steady-state levels, there is great confidence in the data and the validity of the study as the calculated kinetic BCF of 20 agrees well with both the steady-state BCF of 13 from the definitive study as well as the kinetic BCF of 10 determined from pilot study data. As all bioconcentration factors calculated for ZDEC are two orders of magnitude below the threshold values of 1000 and 2000 established by the United States Environmental Protection Agency and the European Chemicals Agency, respectively, ZDEC does not meet the criteria for a “B” designation. As such, repeating the study would not be prudent as it would require further animal use to obtain similar results that would indicate that the test substance does not significantly bioaccumulate in the test organism.