Registration Dossier

Administrative data

Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 1991 - July 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 305 E (Bioaccumulation: Flow-through Fish Test)
Qualifier:
according to
Guideline:
EPA OPP 165-4 (Laboratory Studies of Pesticide Accumulation in Fish)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Radiolabelling:
yes

Test organisms

Test organisms (species):
Lepomis macrochirus
Details on test organisms:
The Bluegill (Lepomis macrochirus) was selected as the test species for this study

Bluegill cultures were held in water from the same source and the same temperature as used during the test. Bluegill were held for 14 days prior to testing and all fish appeared healthy. The fish were acclimated to test conditions for approximately 53 hr before the start of the test. During acclimation, <1% of the fih died. Surviving fish showed no signs of disease or stress.

Fish were from the same source and year class. The total length of the fish, collected during the test, was 52 ± 6.46 mm (mean ± SD), and the range was 39 to 74 mm long. The weight of the fish, collected during the test, was 1.82 ± 0.82 g, and the range was 0.51 to 5.64 g. Loading, defined as the total wet weight of fish per liter of test water that passed through any given test chamber in 24 hr, did not exceed 3.1g of fish/L, did not exceed 0.48 g of fish/L. Instantaneous loading, representing the biomass of fish per liter of test water at given time, did not exceed 3.1 of fish/L.

During the holding, acclimation, and test periods, the bluegill were fed flaked fish food. During the test, excess food was siphoned from the test chambers approximately 30 min after feeding. Feeding and sampling schedules were coordinated, so the digestive tracts of the fish could clear for approximately 24 hr before sampling.

The uptake phase of the test lasted 28 days, after that fish were exposed to untreated well water for a period of 14 days.
In this study the bioconcentration factor (BCF) and uptake and depuration rate constants of Piperonyl Butoxide in the bluegill were determined.

Study design

Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
28 d
Total depuration duration:
14 d

Test conditions

Hardness:
See the table reproted in the document "TEMPERATURE CONDUCTIVITY, ALKALINITY, HARDNESS, AND pH OF WATER IN THE TEST CHAMBERS" attached in "background material"
Test temperature:
See the table reproted in the document "TEMPERATURE CONDUCTIVITY, ALKALINITY, HARDNESS, AND pH OF WATER IN THE TEST CHAMBERS" attached in "background material"
pH:
See the table reproted in the document "TEMPERATURE CONDUCTIVITY, ALKALINITY, HARDNESS, AND pH OF WATER IN THE TEST CHAMBERS" attached in "background material"
Dissolved oxygen:
See the tables reproted in the document "DISSOLVED OXYGEN CONTENT OF WATER IN THE TEST CHAMBERS" attached in "background material"
TOC:
See the tables reproted in the document "WATER CHARACTERIZATION PRECEDING THE TEST" attached in "background material".
This document reports
Salinity:
The water used for the test: is freshwater.
To see the results of the analyses performed to characterize the well water (e.g. the ionic sodium content) see the tables reported in the document "WATER CHARACTERIZATION PRECEDING THE TEST" attached in "background material".
Details on test conditions:
SYSTEM
A continuous-flow diluter system (Aquatic Technology, Inc., Martinez, CA) was used to deliver test substance and a solvent control to the test chambers. A peristaltic pump (Cole-Parmer Instrument Co., Chicago, IL) was used to deliver the dispensing stock solutions to mixing chambers assigned to the treated and control group.
For a single radiolabelled stock solution the radiolabelled test substance was dissolved in methanol and brought to 100 mL with methanol. The concentration in this primary stock was determined to be 2.53 x 107 dpm/mL and the concentration of PBO was calculated to be 0.17 mg/mL.
The dispensing stock solutions were used to deliver the test substance to the diluter system during the test. An aliquot of the 14C PBO stock solution was combined with the nonradiolabelled test substance and diluted to a concentration of 1.0 mg/mL with calculated specific activities of 0.11 mCi/mmol. Approximately 6.4 volume additions of test water were delivered to the test chambers every 24 h.

CONDITIONS
The nominal concentration of PBO in the treated test chambers was 0.1 ppm, the control chambers received methanol at a nominal concentration of 1.0 mL/L. The dispensing stock solutions were changed weekly in the diluter delivery system. Fluorescent tubes, emitting wavelength similar to natural sunlight (e.g. Chroma 50), provided a photoperiod of 16 hours of light and 8 hours of darkness. Light intensity at the water surface was 340 to 970 lux. Each test chamber (2 treatment and 2 control groups) contained 70 fish. The last four fish assigned to test chambers A for both the treated and the control groups were withheld as the day 0 fish samples.
Nominal and measured concentrations:
0.1 mg/l
Details on estimation of bioconcentration:
Observed BCF (BCF0) values were calculated as the ratio of the test substance concentration in fish tissues (CT) at steady state of the average of the concentrations of test substance in water (Cw) at that time using the following formula
BCF0 = CT/CW
BCF0 values were determined for the days 3, 7, 14 and 21 of the uptake phase and a mean BCF0 was calculated (see the document "OBSERVED BCF in FISH TISSUES" attached in "background material").

The mean predicted bioconcentration factor (BCFB) values for PBO were calculated as the ratio of the uptake rate constant (k1) to the depuration rate constant (k2) using the following formula:
BCFB = k1/k2
The uptake and depuration rate constants were calculated with the BIOFAC© computer program, which uses a water concentration (CW)/tissue concentration (CT) kinetics modes (see the document "PREDICTED BCF and UPTAKE and DEPURATION RATE CONSTANTS" attached in "background material").

Results and discussion

Bioaccumulation factor
Key result
Type:
BCF
Value:
>= 91 - <= 380 dimensionless
Depuration
Elimination:
yes
Details on kinetic parameters:
UPTAKE RATE CONSTANTS: mL/g *day
edible tissue: 100
nonedible tissue: 200
whole fish: 160
(see the table reported in the document "PREDICTED BIOCONCENTRATION FACTORS & UPTAKE & DEPURATION RATE CONSTANTS" attached in "background material")

DEPURATION RATE CONSTANTS: per day
edible tissue: 1.0
nonedible tissue: 0.44
whole fish: 0.55
(see the table reported in the document "PREDICTED BIOCONCENTRATION FACTORS & UPTAKE & DEPURATION RATE CONSTANTS" attached in "background material")

The polar PBO residue fraction accounted for 96 % of the 14C residue in edible tissues. The nonpolar fraction accounted for 3.6 % of the 14C residue in edible tissue. Nonextractable 14C residues were less than the LOQ.
Metabolites:
No metabolites identified
Details on results:
- Mortality of test organisms:
Of the 280 fish exposed during the test, ten fish in the control group and seven fish in the treated group died during the uptake phase. One additional fish died in the treatment group during depuration phase. Post-mortem examinations of the dead fish revealed no atypical causes of death. The mortalities in the treatment group were not considered to be treatment related.

- Behavioural abnormalities:
The number of fish displaying abnormal behaviour in the treated group was similar to that of the solvent control group.

- Other observations:
The polar PBO residue fraction accounted for 96 % of the 14C residue in edible tissues. The nonpolar fraction accounted for 3.6 % of the 14C residue in edible tissue. Nonextractable 14C residues were less than the LOQ.

Any other information on results incl. tables

Bioconcentration factors were 91, 260 and 380 for whole
fish, edible and non-edible portions, respectively.

The depuration rate constants (k2 (day-1)) were 0.55, 1.0
and 0.44 for whole fish, edible and non-edible portions,respectively.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
OECD Guideline 305 E (Bioaccumulation: Flow-through Fish Test)
Conclusions:
-Materials and methods
The bioaccumulation and metabolite identification of PBO was studied using Bluegill sunfish (Lepomis macrochirus). The test fish were maintained under flow-through conditions and exposed to PBO at a nominal concentration of 0.1 ppm for 28 days followed by a 14 days depuration phase. A control group was exposed to the solvent (methanol) used to deliver PBO to the treated group. The treated and the control groups had two replicate test chambers that contained 70 fish in each chamber.
Test aquaria maintained a test solution volume of 45 L and a flow of 288 L (6.4 volume replacements) every 24 hours.
Observations on behaviour of the fish and the physical appearance of the test substance were made daily. Dissolved Oxygen concentration was recorded daily in each test substance aquarium and water temperature and pH were recorded at test initiation and in weekly intervals afterwards. Water chemistry was also measured weekly. Twenty eight days after exposure, the fish were transferred into identical aquaria under identical conditions for a depuration period of 14 days.
To quantify the concentration of [14C] residues in the fish, five fish were collected from the exposure aquarium on days 0, 3, 7, 14, 21 and 28 of exposure and days 1, 3, 7, 10 and 14 of depuration for analysis. Five fish were also removed from the solvent control aquarium on the same days and only those removed on the last day of exposure and depuration were analysed. Five fish were also removed from the culture holding tank at the start of the test to measure background residues. The levels and identities of the parent product and degradates in water and tissues were determined by extraction, chromatographic methods, and liquid scintillation counting (LSC).
(Test guidelines no.: US EPA 165-4)

- Results and discussion
BCFs were determined to be 91 and 380 in edible and non-edible tissues, respectively. The whole body BCF was calculated to be 260. These values were very similar to the predicted bioconcentration factors of 99, 290 and 450 for the same respective matrices, calculated as the ratio of the uptake and depuration rate constants. Depuration of PBO was rapid, exceeding 50 % after two days.

- Conclusion
Validity criteria can be considered as fulfilled.
With a BCF exceeding 100, PBO has the potential to quickly bioaccumulate in aquatic organisms but showed also a rapid clearance in the depuration phase (DT50 = 1.6 days)