1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

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:

31 Mar 2000 to 05 Jul 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)

Version / remarks:

June 1996

GLP compliance:

yes

Radiolabelling:

yes

Details on sampling:

SAMPLING
- Water sampling: Oxygen and temperature were measured at least 3 times, pH and conductivity once a week, directly in the aquaria. For the determination of the remaining physical parameters, specimens were taken from the vessels and analyzed. Water specimens of 10 mL from each test aquarium and the control were taken for analysis of total radioactivity in water at days 0, 1, 2, 3, 7, 10, 14, 17, 21, 24 and 28 of the exposure phase and at days 1, 2, 3, 7, 10 and 14 of the depuration phase. For the identification of the test item, specimens of up to 500 mL were taken at days -4, -1, 0, 1, 3, 7, 14, 17, 21, 24, and 28 of the exposure phase, and at day 1 and 14 of the depuration phase. Specimens from the control tank were taken at days 0 and 28 of the exposure phase and at day I and 14 of the depuration phase. Aliquots of 200 mL of the specimens at these days were passed through a Supelclean LC18 SPE solid phase extraction column,which had been preconditioned with about 5 mL of acetonitrile and about 5 mL of bidistilled water. Specimens were eluted with at least 2 mL of acetonitrile and made up to a specified volume with bidistilled water. Their substance content was determined by HPLC.
- Fish: For analysis of radioactivity in edible and non-edible portions of fish specimens, 4 fish were taken from each test tank at days 0, 1, 3, 7, 14, 17, 21, 24 and 28 of the exposure phase and at days 1, 3, 7, 10 and 14 of the depuration phase and placed into a tricaine solution5 for euthanasia. At day 1 and 28 of the exposure phase and at day 14 of the depuration phase, 4 fish of the control tank were taken for background radioactivity measurements. Thereafter, the fish were dissected into edible (body muscle, skin and skeleton) and non-edible parts (fins, head and organs). The corresponding portions were pooled, placed into glass vials and exactly 43 g of tissue solubilizer (Soluene35 (f) were added. After the complete digesting of the tissues, aliquots of the solutions were submitted to LSC. For the analysis of the amount of parent and lipid content, 16 fish were taken from each test tank at day 21 and 28 of exposure. For the analysis of the lipid content, additional fish specimens (8 at each sampling point) were taken from the control tank at days 1, 28, and 42 of the study.

Vehicle:

no

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

- Preparation of the Stock- and Application solutions: For the preparation of the stock solution I, about 2000 mg of 14C-substance were dissolved in accurately 20 mL of ethanol (= SL1). Thereafter, the amount of radioactive material present was determined by Liquid Scintillation Counting (LSC) to be 477 MBq (= 2005 mg) by using the specific radioactivity of 0.238 MBq/mg of the batch. For the preparation of the stock solution 2, about 2024 mg of the substance were dissolved in accurately 25 mL of ethanol. Thereafter, the amount of the substance present was determined by HPLC-analysis to be 1971.5 mg. For the preparation of the stock solution 3, stock solution 1 was mixed with stock solution 2 and made up with ethanol to exactly 50 mL. The amount of labelled and unlabeled test item was determined by HPLC to be 3955 mg. The amount of radioactive material was determined by LSC to be 468 MBq, resulting in a specific activity of 0.118 MBq/mg test item in stock solution 3. Amounts of 809 µL SL3 for the higher and 80.9 µL SL3 for lower concentration, respectively, were needed for the preparation of 1 L of application solution (deion. water). 1080 mL of application solution were needed for 24 h. Application solutions were prepared for 2-4 days at a time and used up until next renewal. The concentration of active ingredient in these application solutions was determined by LSC.
- Treatment: Volumes of 0.75 mL/min of the application solutions were pumped into the diluter and mixed with exactly 750 mL/min of water, giving final concentrations in exposure water of 0.064 mg/L (C1) and 0.0064 mg/L (C2) in the higher and lower concentration tank, respectively. These exposure concentrations were based on the acute and chronic toxicity of the chemical to the test organism and did not exceed 1% and 0.1% of the 96 h LC50, respectively. The control aquarium continuously received dilution water containing 0.001 mL ethanol/ L dilution water with a flow rate of 750 mL/min.

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Species: Juvenile bluegill sunfish, Lepomis macrochirus
- Number of fish: 100 per test tank and 100 in the control tank
- Size and weight: Fish length, measured at the beginning of the study, was on average 6.1 ± 0.4 cm and the smallest and largest fish were 5.6 and 7.0 cm, respectively (for 60 fish). The average weight of the used fish (day 0) was 3.18 ± 0.6 grams for 300 fish.
- Origin and acclimation: The fish were obtained from a commercial fish hatchery and gradually
acclimated to the test conditions. They were held for at least 14 days prior to testing in water of similar quality as used in the study at the required test temperature.
- Mortality: Less than 5% mortality was observed during the acclimatization period and no mortality occurred during the course of exposure and depuration.
- Feeding: The fish were fed commercially prepared discus fish food on working days prior to and during the bioconcentration study. The feeding rate was 2% of the total biomass daily. The fish were not fed 24 hours prior to each sampling.
- Handling: All transfers of fish were performed taking care to minimize possible stress due to the handling. No fish were damaged or dropped during transfer. Loading The fish biomass to water ratio did not exceed 0.3 gram per liter passing through the test system during 24 hours.

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

other: dechlorinated tap water

Total exposure / uptake duration:

28 d

Total depuration duration:

14 d

Hardness:

181 mg/L (as CaCO3)

Test temperature:

22.2 ± 0.13 °C

pH:

7.2 / 7.3

Dissolved oxygen:

7.9- 7.8 mg/L

TOC:

0.5 mg/L

Conductivity:

460 - 460 µS/cm

Details on test conditions:

TEST PROCEDURE
- Procedure: Flow-through aquatic system
- Duration: 28 days for accumulation and 14 days for depuration.
- Test Chambers: Three 153 liter clear glass aquaria were used as test chambers. The water depth was maintained at 40 cm. The water volume in each tank was 128 L.
- Diluter: A continuous flow chemical delivery system was used. The flow of the system was adjusted to 1080 L per day and aquarium during exposure (45 L per hour, corresponding to about 8.44 aquarium volumes per day). One aquarium served as control and received water with 0.001 mL ethanol per L dilution water, the other two aquaria continuously received the 14C-Iabelled test item from the application solution (also containing ethanol). Dosing pumps were used to deliver the appropriate amount of the application solutions into a mixing flask where it was diluted with water to produce the test solution. During the depuration phase, only dilution water (without ethanol) was used for all three aquaria. The flow of the system was the same as during the exposure phase.
- System Calibration and Control: The calibration of the diluter system was checked prior to test initiation. The flow rates of the dosing pumps for both, the dilution water and the application solutions were controlled at least at each working day.
- Water Quality: The water was specified for physical parameters as total hardness, total alkalinity and conductivity before the start of the experiment (Table 1 in “Any other information on materials and methods incl tables”).
- Physical Parameters: Temperature, pH, oxygen, conductivity, hardness and total organic carbon were measured in the test vessels during the study.
- Cleaning: The test aquaria were cleaned by standard laboratory procedures to remove any residual radioactivity before the study was started. During the study, the test aquaria were cleaned at least after each feeding by siphoning out accumulated debris and wiping off the inner glass walls.
- Illumination: Fluorescent light, 16 hours daily with 30 minute transition period.
- Treatment: Volumes of 0.75 mL/min of the application solutions were pumped into the diluter and mixed with exactly 750 mL/min of water, giving final concentrations in exposure water of 0.064 mg/L (C1) and 0.0064 mg/L (C2) in the higher and lower concentration tank, respectively. These exposure concentrations were based on the acute and chronic toxicity of the chemical to the test organism and did not exceed 1% and 0.1% of the 96 h LC50, respectively. The control aquarium continuously received dilution water containing 0.001 mL ethanol/ L dilution water with a flow rate of 750 mL/min.
- Storage of Specimens: Specimens not directly needed for analysis were stored at -18°C in the dark. If specimens were stored before analysis, stability data were collected to show, that the subsequent analysis are valid. At the completion of the study all biological and analytical specimens were destroyed, as their storage stability cannot be guaranteed for longer time periods.

Nominal and measured concentrations:

Nominal concentration: 0.064 mg/L and 0.0064 mg/L
Measured concnetration: Average of 0.0646 ± 0.00192 mg equiv./L for 0.064 mg/L (ranged from 0.0628 to 0.0683 mg equiv./L) and average of 0.00666 ± 0.00013 mg equiv./L (ranged from 0.00638 to 0.00685 mg equiv./L) for 0.0064 mg/L are the concentration of the substance equivalents in water during the accumulation phase.

Reference substance (positive control):

no

Lipid content:

>= 1.1 - <= 2.4 %

Time point:

other: day 1 and day 42 respectively

Remarks on result:

other: % weight of the edible parts for all, control fish, low dose fish and high dose fish

Lipid content:

>= 3.8 - <= 7.6 %

Time point:

other: day 1 and day 42 respectively

Remarks on result:

other: % weight of the non-edible parts for all, control fish, low dose fish and high dose fish

Lipid content:

>= 3.8 - <= 7.6 %

Time point:

other: day 1 and day 42 respectively

Remarks on result:

other: % weight of the whole fish for all, control fish, low dose fish and high dose fish

Key result

Temp.:

22 °C

pH:

8.1

Type:

BCF

Value:

180 dimensionless

Basis:

whole body w.w.

Time of plateau:

17 d

Calculation basis:

steady state

Remarks:

from day 17 to day 28

Remarks on result:

other: mean calculated value

Conc. / dose:

0.064 mg/L

Temp.:

22 °C

pH:

8.1

Type:

BCF

Value:

176 dimensionless

Basis:

whole body w.w.

Time of plateau:

17 d

Calculation basis:

steady state

Remarks:

from day 17 to day 28

Remarks on result:

other: high test concentration

Conc. / dose:

0.006 mg/L

Temp.:

22 °C

pH:

8.1

Type:

BCF

Value:

184 dimensionless

Basis:

whole body w.w.

Time of plateau:

17 d

Calculation basis:

steady state

Remarks:

from day 17 to day 28

Remarks on result:

other: low test concentration

Parameter:

DT50

Depuration time (DT):

0.39 d

Remarks on result:

other: Mean from both concentrations

Parameter:

DT90

Depuration time (DT):

1.29 d

Remarks on result:

other: Mean from both concentrations

Parameter:

DT50

Depuration time (DT):

0.48 d

Remarks on result:

other: Nominal concentration: 0.0064 mg/L

Parameter:

DT50

Depuration time (DT):

0.29 d

Remarks on result:

other: Nominal concnetration: 0.064 mg/L

Details on kinetic parameters:

- Uptake rate constant (Ku): 406/day for the whole fish at 0.064 mg/L
- Depuration rate constant (Kd): 2.354/day for the whole fish at 0.064 mg/L
- Uptake rate constant (Ku): 274/day for the whole fish at 0.0064 mg/L
- Depuration rate constant (Kd): 1.44/day for the whole fish at 0.0064 mg/L
- Calculated bioconcentration factor (BCFk): 39 for whole fishas mean of the two concentrations
- Calculated bioconcentration factor (BCFk): 13 for edible tissues and 62 for non-edible tissues as mean of the two concentrations

Metabolites:

- The amount of the unchanged parent molecule was determined in edible and non-edible fish parts during accumulation (day 21) and at the end of the exposure phase (day 28) for both test concentrations. The substance was a major residue of the edible and non-edible parts of the fish. In the edible part of fish exposed to 0.064 mg substance/L water the concentration of the substance was 1.03 ppm (53.6% of sample) and 1.45 ppm (65%) at days 21 and 28 of exposure, respectively. In the edible part of fish exposed to 0.0064 mg substance/L water the concentration of the substance was 0.07 ppm (34.6%) and 0.09 ppm (36.5%) at days 21 and 28, respectively. The concentration of the substance in the non-edible part of fish exposed to 0.064 mg substance/L water ranged from 3.77 ppm (18.1%) to 5.75 ppm (67.5%) at days 21 and 28, respectively. The concentration of the substance in the non-edible part of fish exposed to 0.0064 mg substance/L water ranged from 0.44 ppm (24.5%) and 0.28 ppm (17.7%) at days 21 and 28, respectively.

Details on results:

WATER QUALITY DATA
During the accumulation and depuration phases, temperature, pH, oxygen concentration and conductivity as well as TOC and hardness, were monitored.

PURITY AND STABILITY OF THE TEST ITEM
- Purity of the Stock Solutions: An aliquot of the stock solution 1 (SL 1) of 14C-substance was submitted to HPLC analysis 7 days before start of the accumulation phase. No impurities (UV-LOQ = 0.04 mg/L) were detected with, using UV/VIS-detection; the content of the test item was 101% of the nominal concentration (100250 mg/L). The analysis of the radio signal (C14-LOQ = 800 dpm/injection) showed a radiochemical purity of > 98%. An aliquot of the stock solution 2 (SL2) of unlabelled substance was submitted to HPLC-analysis 7 days before start of the accumulation. No impurities ( LOQ = 0.04 mg/L) were detected; the content of the test item was 97% of the nominal concentration (see APPENDIX H). An aliquot of the stock solution 3 (SL3 = mixture of SL1 and SL2) was submitted to HPLC-analysis 6 days before start of the accumulation phase. No impurities (UV-LOQ = 0.04 mg/L) were detected using UV/VIS detection; the content of the test item was 98% of the nominal concentration (80580 mg/L). The analysis of the radio signal (C14-LOQ = 800 dpm/injection) showed a radiochemical purity of > 98%. The 2D- TLC chromatogram showed a radiochemical purity of > 99% 7 days before start of accumulation. The new calculated specific activity of the test item in the stock solution 3 was 0.118 MBq/mg.

STABILITY
- Stock Solution: An aliquot of the stock solution 3 (SL3) of 14C-substance/substance was submitted to HPLC- and two dimensional thin layer chromatography (2D-TLC) 41 and 39 days after the end of the depuration phase, respectively. The analysis of the HPLC radio signal showed a radiochemical purity of > 99% and the 2D-TLC chromatogram showed a radiochemical purity of > 98%.
- Application Solution: Aliquots from a freshly prepared application solutions for C1 , day 10, room temperature, were analysed by HPLC. No impurities (UV-LOQ = 0.04 mg/L; C14-LOQ = 800 dpm) were found with radiochemical- and UV/VIS-detection . An additional aliquots from the same application solutions was taken after 4 days at room temperature (day 14) and reanalysed. The HPLC chromatograms showed no change in purity compared to the initial analysis.
- Test Specimen: A day 3 blank specimen was spiked with the test item, creating a concentration of 0.0791 g/L. After storage of the specimen at -20°C for 88 days, no impurities (LOQ = 0.04 mg/L) in the HPLC chromatogram with UV/VIS was observed.

RADIOACTIVITY IN WATER
- Distribution of Radioactivity in Water: The concentration of the substance equivalents in water during the accumulation phase ranged from 0.0628 to 0.0683 mg equiv./L with an average of 0.0646 ± 0.00192 mg equiv./L for the higher concentration (C1). For the lower concentration (C2) the concentration of substance ranged from 0.00638 to 0.00685 mg equiv./L with an average of 0.00666 ± 0.00013 mg equiv./L. The levels of radioactivity measured in the first 2 days of depuration rapidly dropped to <100 ng equiv./L for the higher and to < 1 ng equiv./L for the lower concentration, respectively.
- Identification of Substances in Water: The nature of the test item substance and its concentrations in water specimens of the treated tanks was demonstrated by HPLC-analysis at sampling days -4, - 1 , 0, 1, 3, 7, 14, 17, 21, 24 and 28 of the exposure phase. The HPLC-chromatograms of the test specimens showed no interference with other peaks. The identity
of the parent molecule was proven by co-chromatography. The composition of water specimens was also tested by two dimensional thin layer chromatography (2DTLC)-analysis for Cl at sampling days 21 and 28. The content of 14C-substance in both specimens was 97% in relation to the total radiolabelled material in the specimen.
- Distribution of Radioactivity in Fish: Based on the total radioactivity found in the different fish tissues, the kinetic data were evaluated using a steady-state approach. The substance’s residues were rapidly concentrated in edible and non-edible portions of the fish as well as in the whole fish. The concentrations reached a constant plateau after approximately 17 days for both, the higher (C1) and the lower concentration (C2). At steady state, the concentration of the test item in the whole fish was within ± 20% of the mean steady state concentration for at least 4 successive specimens at intervals of at least 3 days for both, the higher (C1) and the lower concentration (C2). At steady state, the measured concentrations of the substance equivalents in the non-edible and edible tissues and in the whole fish were 20.39, 2.02 and 11.59 mg equivalents /kg tissue fresh weight for the higher concentration (C1) and 2.237, 0.188 and 1.227 mg equivalents/kg tissue fresh weight for the lower concentration (C2), respectively. In the substance’s free water, substance’s residues were rapidly eliminated from the non-edible and edible tissues and the whole fish with DT-90 values of 1.0, 0.79 and 0.98 days for the higher concentration (C1) and 1.63, 1.53 and 1.60 days for the lower concentration (C2). The data of the exposure- and depuration phase indicate, that, under constant exposure, the substance will be bioconcentrated mainly in non-edible fish tissues. Elimination of the bioconcentrated residues will be 99% after 3 days in the substance free water.

LIPID DETERMINATION
The lipid content of the fish was determined gravimetrically after solvent extraction of the edible and nonedible tissues at various time points. The lipid amount of the whole fish was calculated from the results of the edible and non-edible parts. The lipid content (% of weight) of the edible parts was within the range of 1.1% (day 1, control) to 2.4% (day 42, control) for all, control fish, low dose fish and high dose fish. The lipid content of the non-edible parts was within the range of 3.8% (day 1, control) to 7.6% (day 42, control) for all, control fish, low dose fish and high dose fish. The calculated lipid content (% of weight) of total fish at days 1, 21, 28 and 42, was within the range of 2.48% - 4.91% for control fish, 3.03% - 3.31% for high dose (C1) fish, and 3.66% - 4.18% for low dose fish (C2), respectively.

STORAGE STABILITY
- To test the storage stability, day 1 blank edible and non-edible parts of fish were spiked with [Triazole-(U)-14C] substance, extracted and subsequently analysed by 1D-TLC and C-18 HPLC. These extracts were stored at -18°C until the end of the metabolism phase of the study (day 48) and analysed by 1d-TLC and C-18 HPLC. Additionally, spiked fish specimens were stored frozen until the end of the metabolism phase (day 44) and were extracted and analyzed by 1d-TLC and C-18 HPLC. The substance was stable in the fish extracts during the 44/48 days freezer storage period. Since all fish specimens of the study were extracted within 3 days of sampling and analyzed within 13 days after extraction, the parent results are valid.

ESTIMATION OF BIOCONCENTRATION FACTORS
- Bioconcentration factors (BCFs) were determined by calculating the ratio of the steady-state concentration of the substance equivalents in fish tissues and whole fish to the average concentration of the substance in water during steady-state (measured BCF, based on total radioactivity). In addition, bioconcentration factors for the same tissues were estimated calculating the ratio of the uptake rate constant to the depuration rate constant (calculated BCF). A summary of the measured and calculated bioconcentration factors is given in Table 1 in “Any other information on results incl tables”. The measured bioconcentration factors for the substance equivalent residues in non-edible portions, edible portions and the whole fish were 309, 31 and 176 for the higher, and 335, 28 and 184 for the lower concentration, respectively. Thus, the mean measured BCF for the substance is 180 for the whole fish. The calculated BCFs from the kinetic data for the same tissues were 310, 27 and 172 for the higher concentration (C1) and 353, 27 and 190 for the lower concentration (C2), respectively, giving a mean calculated BCF of 181 for the whole fish. Bioconcentration factors, calculated for parent were based on the metabolite pattern in fish tissues at days 21 and 28 of the accumulation as seen in Table 1 in “Any other information on results incl tables”. The bioconcentration factors for parent at day 21 in non-edible portions, edible portions and the whole fish were 57, 16 and 38, for the higher (C1), and 66, II and 40 for the lower concentration (C2), respectively. The bioconcentration factors for parent at day 28 in non-edible portions, edible portions and the whole fish were 87, 22 and 54, for the higher (C1), and 43, 13 and 28 for the lower concentration (C2), respectively. The change of bioconcentration factors with time and its difference at day 28 for C1 and C2 was probably due to ongoing metabolic processes which are both, time- and concentration dependent. According to the correlation estimated by Mackay (1982), the predicted BCF value for a substance with a log Kow of 3.72 is 25112. The course of the accumulation and depuration and the BCF’s determined in the present experiment indicate only a limited bioconcentration of the substance in the bluegill, Lepomis macrochirus.

VALIDITY OF THE STUDY

- The temperature variation during the accumulation and depuration phases was less than ± 1 °C with 1 exception at day 8 of the study, where the temperature was at 16.5°C for 6 hours in all vessels (mean of values according to Table 3: 22.2 °C ; mean standard deviation: 0.13 °C; minimum: 21.9 °C; maximum; 22.8 °C).

- The concentration of dissolved oxygen did not fall below 60% saturation during the whole study period (mean: 90 %; mean standard deviation: 2.51; minimum: 78%; maximum: 99%).

- The concentrations of the test item (based on total radioactivity) in the water of the test aquaria were maintained within ± 20% of the mean of the measured values during the accumulation phase. The actual ranges were 97% - 106% for the higher and 96% - 103% for the lower test concentration.

- 7 days prior to exposure, the mortality of the fish was below 5% and no fish died during the accumulation and depuration phases.

- The fish showed no sublethal symptoms during the accumulation and depuration phases.

- There were no circumstances that affected the quality and integrity of the data.

Table 1. Bioconcentration factors

Nominal concentration: 0.064 mg/L | Steady state from day 17 to day 28
Average Concentration in Edible Portions		2.02 [mg equiv./kg]
Average Concentration in Non-Edible Portions		20.39 [mg equiv./kg]
Average Concentration in Fish		11.59 [mg equiv./kg]
Average Concentration in Water (days 17-28)		0.0659 [mg equiv./kg]
Bioconcentration Factors based on substance Equivalents
	Edibles	Non-Edibles	Total
Measured (Ctissue/Cwater)	31	309	176
Calculated (ku/kd)	27	310	172
Bioconcentration Factors calculated for Parent Substance
	Edibles	Non-Edibles	Total
BCFparent day 21	16	57	38
BCFparent day 28	22	87	54
Nominal concentration: 0.0064 mg/L | Steady state from day 17 to day 28
Average Concentration in Edible Portions		0.188 [mg equiv./kg]
Average Concentration in Non-Edible Portions		2.237 [mg equiv./kg]
Average Concentration in Fish		1.227 [mg equiv./kg]
Average Concentration in Water (days 17-28)		0.00667 [mg equiv./kg]
Bioconcentration Factors based on substance Equivalents
	Edibles	Non-Edibles	Total
Measured (Ctissue/Cwater)	28	335	184
Calculated (ku/kd)	27	353	190
Bioconcentration Factors calculated for Parent Substance
	Edibles	Non-Edibles	Total
BCFparent day 21	11	66	40
BCFparent day 28	13	43	28
Mean from both concentrations
Bioconcentration Factors based on substance Equivalents
	Edibles	Non-Edibles	Total
Measured (Ctissue/Cwater)	30	322	180
Calculated (ku/kd)	27	332	181
Bioconcentration Factors based on substance Equivalents, day 21
	Edibles	Non-Edibles	Total
BCFparent	13	62	39

 

Validity criteria fulfilled:

yes

Remarks:

see 'Any other information on results incl. tables'

Conclusions:

The substance residues were rapidly concentrated in fish tissues, reaching a stable steady-state concentration within about 17 days for the higher and the lower concentration. Measured bioconcentration factors (as a mean of the lower and the higher concentration) for the substance’s residues in non-edible portions, edible portions and whole fish were 322, 30 and 180, respectively. The calculated BCFs (as a mean of the lower and the higher concentration, again) for the same tissues were 332, 27 and 181, respectively. The depuration time for 90% of the bioconcentrated radioactivity for the whole fish was less than 1 day for the higher and less than 2 days for the lower concentration, respectively. In conclusion, the substance showed only limited bioconcentration in bluegill, Lepomis macrochirus, and bioconcentrated residues were rapidly eliminated in the substance free water.

Executive summary:

The bioaccumulation of the substance in fish was performed according to the OECD TG 305 and in compliance with GLP criteria. The substance has a log Kow (Pow) of 3.72. According to the correlation estimated by Mackay (1982), the predicted bioconcentration factor (BCF) in fish for a compound with that Kow value is 251. Information on the uptake of the substance from water and its depuration from fish is useful in assessing the relative propensity of the test item to enter and persist in aquatic food chains. The uptake and depuration study was conducted with the bluegill sunfish,Lepomis macrochirus. The objectives were to determine the bioconcentration factor, the time to reach steady-state concentration in fish tissues, the amount of parent and lipid content in edible and non-edible fish portions and the accumulation and depuration rates of the substance in these fish tissues. For this purpose, fish were continuously exposed to 14C-triazole-labelled substance at a concentration of 0.064 mg/L (C1) and 0.0064 mg/L (C2), respectively, for 28 days in a dynamic flow-through system. Thereafter, the depuration of radioactivity in untreated water was initiated. The test concentrations were chosen to be 100 and 1000 times lower than the acute toxicity (LC50) of the substance to bluegill sunfish (Lepomis macrochirus). The substance residues were rapidly concentrated in the edible and non-edible portions of the fish, reaching a constant plateau after 17 days for the higher and the lower concentration. At steady state, the measured concentrations of the substance equivalents in the non-edible and edible tissues and in the whole fish were 20.39, 2.02 and 11.59 mg equivalents/kg tissue fresh weight for the higher concentration and 2.237, 0.188 and 1.227 mg equivalents/kg tissue fresh weight for the lower concentration, respectively. The measured bioconcentration factors for the substance equivalent residues in non-edible portions, edible portions and the whole fish were 309, 31 and 176 for the higher, and 335, 28 and 184 for the lower concentration, respectively. Thus, the mean measured BCF for the substance is 180 for the whole fish. The calculated BCFs from the kinetic data for the same tissues were 310, 27 and 172 for the higher concentration (C1) and 353, 27 and 190 for the lower concentration (C2), respectively, giving a mean calculated BCF of 181 for the whole fish. The amount of the unchanged parent molecule was determined in edible and non-edible fish parts during accumulation (day 21) and at the end of the exposure phase (day 28) for both test concentrations. The substance was a major residue of the edible and non-edible parts of the fish. In the edible part of fish exposed to 0.064 mg substance/L water the concentration of the substance was 1.03 ppm (53.6% of sample) and 1.45 ppm (65%) at days 21 and 28 of exposure, respectively. In the edible part of fish exposed to 0.0064 mg substance/L water the concentration of the substance was 0.07 ppm (34.6%) and 0.09 ppm (36.5%) at days 21 and 28, respectively. The concentration of the substance in the non-edible part of fish exposed to 0.064 mg substance/L water ranged from 3.77 ppm ( 18.1%) to 5.75 ppm (67.5%) at days 21 and 28, respectively. The concentration of the substance in the non-edible part of fish exposed to 0.0064 mg substance/L water ranged from 0.44 ppm (24.5%) and 0.28 ppm (17.7%) at days 21 and 28, respectively. Bioconcentration factors, calculated for parent were based on the parent concentration in fish tissues at days 21 and 28 of the accumulation. The bioconcentration factors for parent at day 21 in non-edible portions, edible portions and the whole fish were 57, 16 and 38, for the higher (C1), and 66, 11 and 40 for the lower concentration (C2), respectively. The bioconcentration factors for parent at day 28 in non-edible portions, edible portions and the whole fish were 87, 22 and 54, for the higher (C1), and 43, 13 and 28 for the lower concentration (C2), respectively. The change of bioconcentration factors with time and its difference at day 28 for C1 and C2 was probably due to ongoing metabolic processes which are both, time- and concentration dependent. After termination of exposure, substance’s residues were rapidly eliminated from the non-edible and edible tissues and the whole fish with DT-90 values of 1.0, 0.79 and 0.98 days for the higher concentration (C1) and 1.63, 1.53 and 1.60 days for the lower concentration (C2). In conclusion, the substance showed only limited bioconcentration in bluegill, Lepomis macrochirus, and elimination of the bioconcentrated residues was 99% after 3 days in the substance free water.

Description of key information

All available data were assessed, and the study representing the worst-case effect was included as the key study. Other studies are included as supporting information. 

BCF = 180 L/kg in whole fish (bluegill sunfish), OECD TG 305, Volz 2000

Key value for chemical safety assessment

BCF (aquatic species):

180 L/kg ww

Additional information

Three standard guidelines followed GLP studies are available for this endpoint (one Reliability 1 and two Reliability 2 studies). The study representing the worst-case effect (i.e. showed the highest BCF value) was included as the key study. The effect value (BCF = 180) from the key study was selected in CSA. In this study (Volz 2000), bluegill sunfish (Lepomis macrochirus) were continuously exposed to the 14C-triazole-labelled substance at a concentration of 0.064 mg/L (C1) and 0.0064 mg/L (C2) for 28 days in a dynamic flow-through system. Thereafter, the depuration of radioactivity in untreated water was initiated. The mean measured BCF was determined to be 180 for the whole fish. In the two supporting studies, the BCF was found to be 116X (LeBlanc & Mastone 1980) and a maximum of 28 L/kg (Thompson & Cranor 1982) in the whole body.