spirodiclofen (ISO);3-(2,4-dichlorophenyl)-2-oxo-1-oxaspiro[4.5]dec-3-en-4-yl 2,2-dimethylbutyrate

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

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 72-6 (Aquatic Organism Accumulation Tests)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 165-4 (Laboratory Studies of Pesticide Accumulation in Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Details on sampling:

Fish sampling details:
Schedule - Fish samples on Day 0, 1, 3, 7, 10, 14, 21 and 29 during exposure and on Day 30, 31, 35, 38 and 42 of depuration.
Number of fish - 4 fish from each treatment level on each sample day. Fish were processed individually.
Dissection details - Dissection into two part: edible (fillet = body, muscle, skin and skeleton) and non-edible (viscera = head, fins, internal organs).
Lipid content of fish - Lipid content was measured on Day 0 and Day 42. 4 fish each day to determine mean.

Processing fish samples:
Sample procedures - Samples were transferred into pre-weighed polystyrene vials. Recorded wet weight of the samples. Then they were frozen, lyophilized, re-weighed, and homogenized. Aliquots were analyzed for radioactivity measurement. Aliquots were also used for determination of metabolites in fish tissues.
Timing of sampling processing - Combustion and measurement of total radioactivity of fish samples was completed on the same time schedule as the test solution analyses. Fish samples were normally analyzed in triplicate if enough tissue was available

Recovery of chemical: 3 test solution samples were taken from each treatment level on Day 0, 1, 3, 7, 10, 14, 21 and 29 of exposure phase and on Day 30, 31, 35, 38 and 42 of depuration phase. Stock was analyzed on Day -4 and Day 29. All samples were analyzed for total radioactivity and percent parent.

Vehicle:

yes

Remarks:

acidified methanol (2.5% acetic acid). The solvent (vehicle) was acidified to increase the stability of spirodiclofen in the stock solution. Acidification did not appear to have an adverse affect on the fish or the pH of the test solutions.

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

Details not provided in summary.

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name: Bluegill sunfish
- Body weight/length: mean body weight of 2.3 g and a mean body length of 5.5 cm.

ACCLIMATION
- Acclimation period: Fish were acclimated to test conditions for more than 1 month. Feeding rate was 1% of mean body weight with standard fish feed. Less than 5% mortality was noted in the 14 days prior to test initiation. 60 randomly selected bluegill in each treatment level.

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

29 d

Total depuration duration:

13 d

Hardness:

Not provided

Test temperature:

21 to 22°C

pH:

6.5 to 7.7

Dissolved oxygen:

83% to 106% saturation

TOC:

Not provided

Salinity:

N/A

Conductivity:

Not provided

Details on test conditions:

Test conditions:
Static renewal/flow through - flow through rate for 6 volume turnovers per day.
Type of dilution system for flow through method - The diluter system toxicant delivery system and flow meters were calibrated.
Aeration - batch water was aerated. No mention of aeration in the aquaria. Duration of the test - Total of 42 days. 29 day exposure to spirodiclofen, 13 day post-treatment exposure to clean water.

Test vessel:
Type/size - Glass aquaria, approximately 100 liter test solution volume.
Biomass loading - Initial loading was 1.29 g fish/L and 0.21 g fish/L/day.
Source of dilution water - reconstituted water.

Nominal and measured concentrations:

Nominal: solvent control (100 µl/L dilution water), 2 µg/L and 20 µg/L.
Measured: 1.6 µg/L and 11.4 µg/L.

Reference substance (positive control):

no

Lipid content:

6 %

Time point:

end of exposure

Remarks on result:

other: Based on BCF parent (whole fish)

Key result

Conc. / dose:

20 µg/L

Temp.:

21.4 °C

pH:

7

Type:

BCF

Value:

1.8 dimensionless

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: Based on Total Radioactive Residues (trr)

Key result

Conc. / dose:

20 µg/L

Temp.:

21.4

pH:

7

Type:

BCF

Value:

1.4 dimensionless

Calculation basis:

steady state

Remarks on result:

other: normalised to 6% lipid content

Reported statistics:

The uptake rate constant (K1) and depuration rate constant (K2) were determined by the Dow BIOFAC computer program (2). This is a non-linear kinetic modeling program which provided optional parameter estimates of rate constants K1 and K2 by utilizing the actual (observed) bioconcentration study data. Preliminary values for K1 and K2 were calculated according to OECD 305 (Annex 4) (11). The bioconcentration factor at steady-state, the time to reach 90 % of steady-state for total 14C-residues in edible parts of fish, non-edible parts of fish and in whole fish, and the time to reach 1/2 of test compound clearance (depuration) were also calculated from the estimated rate constants. A measure of the variability of the estimated parameters were provided by the standard deviation of each estimate.

Sub-lethal tox and other effects: The bluegill showed no mortalities or abnormal behavior throughout the test in all test concentrations.

 

Study deficiencies:

Methanol acidified with a low percent glacial acetic acid was used as the solvent. While methanol was an approved solvent, the addition of the glacial acetic acid was not. However, the slight acidification of the solvent (stock solution) was used to increase the analytical recovery in the test solutions. The percent acid in the solvent was low (2.5%), and the percent acid in the test system, given that the solvent load was 0.1 mL/L, was only 0.00025%. This low amount of acid in the solvent did not cause an appreciative shift in pH, and had no impact on the health of the study organisms, as shown by the solvent control data. Glacial acetic acid was not intrinsically toxic to fish as shown by Yoshida & Nishiuchi (1976). Therefore, the use of acidified methanol as a solvent was a minor deficiency and had no impact on the validity of the study.

 

Reviewers comments:

Storage stability was not specifically addressed in this bioconcentration study. However, the spirodiclofen data package does support the findings in this study. The spirodiclofen parent was not found to any significant degree in the bluegill tissues and this is consistent with other animal metabolism findings and the behavior of parent compound in other matrices such as soil and water. Stability was demonstrated in the goat metabolism samples. The first samples were analyzed within 2 weeks of exposure and again 5 months later. In these tissues the parent was not present to any significant degree. Based upon the extensive research submitted on spirodiclofen in various natural systems, such as soil, water and animal matrices, it is clear that hydroxylation to the enol is rapid and consistent. Therefore, the lack of parent compound in the edible and non-edible tissues of the bluegill is a reasonable result. Based upon the weight of evidence, it is clear that the parent was not detected due to the fish metabolism and was not an artifact of sample storage. The stability of the main metabolites is supported in the referenced reports and is sufficient to verify the bioconcentration samples stability and integrity.

 

Table 2: Calculation results for bioconcentration of spirodiclofen in bluegill.

Calculation results	2 μg/L	2 μg/L	2 μg/L	20 μg/L	20 μg/L	20 μg/L
Fish fraction	Edible	Viscera	Whole fish	Edible	Viscera	Whole fish
Bioconcentration Factor (BCF trr)	131 (± 70.1)	1091 (± 494)	537 (± 129)	114 (± 16.6)	1067 (± 269)	521 (± 127)
Time to reach 90% of steady state	1.98 (± 0.79)	3.03 (± 0.93)	2.62 (± 0.42)	6.03 (± 0.37)	2.13 (± 0.36)	2.20 ( ± 0.34)
t(1/2) for clearance in days	0.60 (± 0.24)	0.91 (± 0.28)	0.79 (± 0.13)	1.82 (± 0.11)	0.64 (± 0.11)	0.66 (± 0.10)
(K1) Uptake rate constant (L/day)	152 (± 54.5)	829 (± 276)	472 (± 84.5)	43.7 (± 5.73)	1155 (± 215)	544 (± 101)
(K2) Clearance Rate constant (L/day)	1.16 (± 0.46)	0.76 (± 0.23)	0.88 (± 0.14)	0.38 (± 0.02)	1.08 (± 0.18)	1.04 (± 0.16)

 

Validity criteria fulfilled:

yes

Conclusions:

Summarizing all findings in fish and water, it can be concluded that both spirodiclofen and its major metabolites in fish do not accumulate (do not bioconcentrate) in fish. Metabolism of spirodiclofen in fish and excretion of its major metabolites back into the water stream are strong and rapid. The bioconcentration factor (wet weight) is 1.8 and the bioconcentration factor normalized to 6% lipid content is 1.4 (both these figures are based on a whole fish).

Executive summary:

Bluegill sunfish (Lepomis macrochirus) were used to determine the spirodiclofen bioconcentration factor in a flow-through dosing system. The fish had a mean body weight of 2.3 g and a mean body length of 5.5 cm. The nominal concentrations were solvent control, 2 and 20 ug 14C-spirodiclofen/L. The exposure period was 29 days. After the exposure, the test fish were exposed to clean dilution water for 13 days, in order to determine the depuration of 14C-spirodiclofen. Biotransformation based on metabolism of spirodiclofen in fish tissues (edible parts and viscera) and depuration of degradates (metabolites) into the test water was examined by identification and quantification of metabolites using radio-TLC and radio-HPLC. The most important metabolites (> 10% of parent or >50 ppb in fish) were identified in fish tissues and also in water using co-chromatography with radiolabelled reference compounds. Lipid was extracted from fish tissues in order to quantify the mean lipid content in the test batches of fish. Summarizing all findings in fish and water, it can be concluded that both spirodiclofen and its major metabolites in fish do not accumulate (bioconcentrate) in fish. Metabolism of spirodiclofen in fish and excretion of its major metabolites back into the water stream are strong and rapid. This toxicity study is classified as acceptable and satisfies the guideline requirement for a bioconcentration study with fish.

Description of key information

The bioconcentration factor (wet weight) is 1.8 and the bioconcentration factor normalized to 6% lipid content is 1.4 (both these figures are based on a whole fish).

Key value for chemical safety assessment

BCF (aquatic species):

1.8 dimensionless