Bumetrizole

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Details on sampling:

After confirmation of stable test solution concentrations, 105 fish were distributed into each test vessel according to a randomization plan generated by the laboratory data evaluation group. The total length and wet body weight of each fish was measured at the start of exposure (study day 0) prior to distribution among the test vessels and again at each sacrifice. Total length (from the top of the snout to the end of the caudal fin) was measured to the nearest
1 mm with a metric ruler and wet weights were measured to the nearest 0.01 g on an analytical balance. Test vessels were observed daily to evaluate mortalities and any individuals exhibiting clinical signs of toxicity or abnormal behavior. Any dead individuals were removed when observed.

Test solution and fish were sampled for radioactivity measurement during the uptake and depuration periods according to the schedule below. Both test solution samples and fish samples were collected concurrently and always prior to first feeding on the sampling day.
Test solution samples (10 mL per sample) were collected directly from the middle of the test vessel. At each sampling time, 5 fish were removed from the test vessel using a net and were sacrificed by immersion in a buffered anesthetic solution (MS222) followed by a brief rinse under running tap water. This procedure also removed residual test solution from the surfaces of the organisms. The fish were blotted dry then weighed and total length measured. Since fish were too large to be combusted whole, they were dissected into at least two parts: edible (fillet; muscle tissue only to the extent possible); and non-edible (remaining fish including: head, gills, bones, skin, fins and internal organs). The parts from each fish were weighed (the total weight of the fish was calculated from the individual parts) and dried separately on filter paper overnight then combusted in an oxidizer and analyzed by liquid scintillation as soon as possible. Since the same number of fish are removed from the control group at the same time, the biological loading (fish/water) in the control and in the test group was kept equivalent.
At each sampling time 5 control fish were sacrificed for radioactivity background correction and to determine lipid content. For background correction, 2 fish were dried and combusted identically as the fish from the concentration group. The lipid content was determined in the remaining 3 control fish which were stored in a freezer until analyzed. The control fish sampled at additional time points were discarded

Sampling time schedule during the uptake period:

Sampling time Test solution samples / concentration No. of fish to sample / concentrationd

Uptake period
Day 0 2 -
Day 3 2 5
Day 7 2a 5
Day 14b 2 (day 15)b 5
Day 21 2 5
Day 28c 2 (day 29)c 5
Day 35 2 5
Day 42 2 5
Day 44 2 5

Depuration period
D.-Day 3 2 5
D.-Day 7 2 5
D.-Day 14 2 5
D.-Day 21 - 5
D.-Day 28 - 5
D.-Day 35 - 5
D.-Day 42 - 5
D.-Day 49 - 5
D.-Day 56 - 5
D.-Day 63 - 5
a = on day 7 measurement of centrifuged samples was forgotten, therefore the mean of all measured centrifuged samples was taken for further calculations as measured concentrations varied within 20% of the mean measured concentration.
b = on day 14 measurement of centrifuged samples was forgotten, therefore the duplicate measurement was repeated on day 15 and since it was comparable to the other results, these values from d15 were used
c = on day 28 only one measurement was conducted instead of two, therefore the next day the duplicate measurement was repeated and since it was comparable to the d28 result, these values from d29 were used
d = from the control group, three fish are used for lipid analyses and two for background correction
- = no samples taken

Vehicle:

yes

Remarks:

The test substance was dissolved in acetone. The concentration of acetone in all test vessels was approx. 0.02 mL/L. The control group received the solvent in the same concentration as the treatment group.

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

TEST SUBSTANCE PREPARATIONS
To achieve the target test concentration, a concentrated stock solution (11.6 mg/L) in acetone was prepared. Approx. 1.16 mg of the [14C] test substance in 0.2283 g Toluene was weighed into a glass vial.
The weighed radiolabeled test substance was stored in a freezer (approx. -18°C or colder) until use, if necessary. The toluene was completely evaporated and afterwards the test substance was re-dissolved in acetone. First, a small volume of acetone was added to dissolve the radiolabeled test substance, then this acetonic solution was added to a glass flask, followed by several acetone rinses of the weighing vessel to insure complete transfer of all test substance. Further acetone was added to the flask to achieve the final target volume. For better dissolution, the acetonic stock solution was ultrasonicated for approx. 30 minutes.
The theoretical concentration and homogeneity were confirmed by the measurement of radioactivity from 3 different regions of the test solution tank in centrifuged and uncentrifuged samples prior to the start of exposure, at study day 3 and once again at steady state (measurements without a GLP status). Since a difference in the radioactivity content was observed between centrifuged and uncentrifuged samples during the concentration verification and homogeneity verification, further analyses and results have to be based on both, centrifuged and uncentrifuged water samples. However, a minimum radioactivity of 500 dpm/ 10 mL test solution is needed to produce reliable results. The results of centrifuged samples were below 500 dpm/ 10 mL and should therefore be taken with care, since they are not fully reliable.

Stock solution preparation:
Nominal test concentration (µg/L) Weight amount of a.i. test substance (mg) Volume of acetone (a) (mL)
0 (sc) - 100
0.2 0.926 100
a to be adjusted depending on weighing of test substance
sc: solvent control

Fresh stock solutions were generally prepared and exchanged on a weekly basis.
The concentration in the stock solution was verified in each newly prepared stock solution without a GLP status. The results were used as the basis to adjust the flow rates of the stock solution pumps and will not be reported in detail.

The in-life exposure was conducted in a continuous flow through system. Metering pumps delivered the stock solution into a mixing vessel where it was instantly mixed with tempered test water (flow controlled by a rotameter) to the nominal test concentrations. The test solution then flew into the test vessels. The flow through system was started 6 days before insertion of the fish to allow equilibration. 
Throughout the exposure period the stock solution was continuously diluted with aerated dilution water at a constant rate (flow controlled by a rotameter) per test group.

Test group Test concentration(nominal) [µg/L] Flow rate of stock solutiona [mL/min] Flow rate of test water [mL/min]
0 0 (sc) 0.006 347
1 0.2 0.006 347
a to be adjusted depending on the measured stock solution concentration. Expected flow rates with a stock solution of 11.6 mg/L (theoretical concentration) and approx. 5-fold tank volume exchange per day.
sc: solvent control

Theoretical radioactivity in the solutions:
1007 dpm / 10 mL in the test solution, 0.2 µg/L
291972 dpm / 50 µL in the stock solution, 11.6 mg/L

The flow rates were calibrated (maximum deviation less than 10%) before the exposure was started and checked weekly during exposure. The flow rates were adjusted during the first days of uptake and depuration and the rate was recorded in the raw data. The required test temperature of 13 ±1°C was ensured by adjusting the test water temperature prior to entering the mixing tanks.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Species: Rainbow trout (Oncorhynchus mykiss). The identity of the species was guaranteed by the supplier.
Origin: Supplier: Forellenzucht Trostadt GmbH & Co. KG, Trostädter Dorfstrasse 7, 98646 Reurieth, Germany
All fish were taken from the same batch of fish of the same origin with the same hatch date.
Sex: Immature male and female fish in unknown sex ratio.
Body size / age at start of exposure: Body weight: overall mean of 0.8 g/fish (±0.2, SD) with a range of 0.7 – 1.0 g/fish. The minimum fish body weight was 70% of the maximum body weight
Body length (overall mean): 4.6 cm ±0.2
Hatch date: 31 May 2019
Arrival in the test facility: 02 Jul 2019
The fish were approximately 3 month old at the start of exposure.
Reason for species selection: Rainbow trout is used routinely for toxicity tests in the testing facility

Husbandry before start of exposure
Acclimation: The fish are maintained for at least 14 days in the laboratory under the same conditions as during the test.
Holding tanks: Prior to testing, the batch of fish were housed in a fiberglass tank (approx. 300 L) receiving a continuous flow through supply of fresh test water.
Photoperiod: 16 hours light, 8 hours dark
Water quality: Non chlorinated charcoal filtered drinking water (Frankenthal, Germany), mixed with deionized water.
Total hardness: Approx. 1 mmol/L = 100 mg/L CaCO3
Acid capacity: Approx. 2.5 mmol/L
Oxygen content: >80% saturation
pH-value: Generally 7.5 – 8.5
Temperature: 13°C
Diet: Commercial fish diet: Inicio 917, 1.1 mm (BioMar, 7330 Brande, Denmark). The composition is suitable for the test species. Information on the composition is provided in the Appendix. Additionally, frozen brine shrimp (Artemia) were provided generally on workdays.
Amount of feed: During acclimation, approx. 1 2% of the mean body weight per day
Medical treatment: The test fish were not treated. The animals were visually inspected for their state of health upon arrival at the test facility and before the start of exposure. Only healthy fish were used.
Mortality during the last week:
none

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason: For comparison to the regulatory BCF threshold values

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

44 d

Total depuration duration:

63 d

Hardness:

Total hardness: Approx. 1 mmol/L = 100 mg/L CaCO3

Test temperature:

12-13 °C

pH:

8.0 – 8.2

Dissolved oxygen:

8.3 – 10.3 mg/L in the test vessels and not less than 60% of the maximum saturation at the test temperature of 13 °C during the test

TOC:

TOC values determined in the control group ranged from 7.8 - 10.5 mg/L during the test period due to the use of acetone as solvent. The TOC values determined in the used water before addition of acetone in the solvent control ranged from 0.6 – 1.4 mg/L. The measured value for hardness in the mixed water supply was at 1.02 mmol/L (approx. 102 mg/L CaCO3) at study day 0.

Salinity:

n/a

Conductivity:

281 µS/cm on day 0

Details on test conditions:

Conditions during exposure
Exposure: Flow through system
On 18 Sep 2019 there was a technical issue with the deionized water supply, which had to be repaired. Therefore, the test system was set to static conditions for 5 hours to guarantee the right composition of the mixed water. Since the duration of this situation was very short, this deviation had no influence on the outcome of the study.
Water temperature: 12-13°C
Aeration: The test water is saturated with oxygen. An aeration in the test vessels was not used.
Photoperiod: 16 hours light, 8 hours darkness
Apparatus: Wherever possible the apparatus used consisted of inert materials like stainless steel and glass. During the test the fish were maintained in silicon-sealed glass aquaria with an overflow at approx. 36 cm, dimensions: 80 x 35 x 55 cm; water volume approx. 100 L.
Flow rate: Approx. 21 L/hour and test vessel resulting in an approx. 5-fold volume exchange per day
Loading: At start of the uptake period 105 fish with a mean body weight of 0.8 g (wet weight) were inserted into each test vessel. Therefore, based on the flow rate of 504 L/d the loading was 0.17 g/L test water per day at start of the uptake period. During the rest of the uptake and depuration period the loading rate was decreased by removing fish from the test vessels for samples.
Test water/mixed water: Aerated non chlorinated charcoal-filtered drinking water from the municipal water works of the city of Frankenthal (67227, Germany), mixed with deionized water prepared in the testing facility. The mixing ratio was adjusted to receive water with a hardness of approximately 100 mg/L CaCO3 and a total organic carbon content of generally ≤2 mg/L. The test water is sanitized by UV treatment prior to entering the aquaria.
The drinking water used to prepare the test water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal (67227, Germany) and the department Environmental Analytics Water / Steam Monitoring of BASF SE as well as for presence of microbes by a contract laboratory. On the basis of the analytical findings, the water was found to be suitable. The German Drinking Water Regulation (Trinkwasserverordnung) served as guideline for maximum tolerable contaminants.
The analytical data for the drinking water used for the preparation of the mixed water are archived with the raw data.
Diet: Same as during adaptation, however no Artemia were provided as food during the test.
Feeding rate: Approx. 1% of the mean body weight per day, generally in 1 portion per day. The amount of food was adapted to the number of fish in the test vessel after each sampling.
Food analysis for contaminants: The food is regularly analyzed for contaminants and the results are stored with the raw data. In view of the aim and duration of this study, the contaminants contained in commercial feed should have no influence on the results.
Cleaning of the test vessels: The test vessels were cleaned daily, generally no sooner than 30 minutes after feeding, to remove residual food and excreta.

TEST GROUPS AND CONCENTRATIONS
Each test group consisted of 1 aquarium with 105 fish randomly inserted at start of test exposure. Each test group aquaria were identified with appropriate labels.

Test group Test substance (TS) concentration in the test solutions No. of fish
TS total (µg/L) [14C]- TS (µg/L) a
0 0 (sc) 0 105
1 0.2 0.1978 105
a: radiochemical purity of test substance = 98.9%
sc: solvent control

The test substance was dissolved in acetone. The concentration of acetone in all test vessels was approx. 0.02 mL/L. The control group received the solvent in the same concentration as the treatment group.
The distribution of fishes to the test groups was done according to a randomization plan prepared by using a program of the laboratory data evaluation group of the testing facility.
In the interest of animal welfare and after agreement of the sponsor, this study used only one concentration of test substance to determine bioconcentration as recommended in the 2012 revised OECD 305 test guideline for non-polar organic chemicals. Recent scientific publications provide compelling evidence that BCF values do not differ when multiple concentrations are tested. The test concentration was well below any known level of toxicity to fish.
 

Nominal and measured concentrations:

nominal: 0.2 µg/L
mean-measured: 0.158 ± 0.022 µg/L. The concentration was kept constant within the range of ±20% of the mean measured concentration throughout the exposure period

Reference substance (positive control):

no

Details on estimation of bioconcentration:

Generally, the bioconcentration factor (BCF) at a specific sampling time was calculated by dividing the concentration in fish at this time, CF(t), by the mean value of the concentration in water (CW) during the uptake period. BCF values in this study were calculated based on the steady state concentration in fish from the plateau phase of the uptake period (BCFss) and based on the uptake and depuration curves by using a first order (one-compartment) biokinetic model (BCFk). The BCF values were further normalized to 5% fish lipid content and corrected for growth during the experiment.
Since in this study a steady state was reached during the uptake period, the steady state bioconcentration factor (BCFss) was calculated according to the following formula:
BCFss = CFss / CWss
Where,
CFss: Steady state concentration of the test substance in fish (µg/kg wet weight), mean of all concentration values in fish of samples taken after steady state was reached.
CWss: Steady state concentration of the test substance in water (µg/L), mean of all concentration values in water.

The kinetic bioconcentration factor (BCFk) was calculated using all measured data from the uptake and depuration curves according to the following formula:
BCFK = k1 / k2
Where,
k1: Uptake rate constant from water (day -1)
k2: Depuration rate constant (day -1)

The uptake and depuration rate constants (k1 and k2) were derived by simultaneously fitting the measured concentrations in fish over time to the following function:
Uptake period: CF (t) / CW = (k1/k2)*(1-e-k2t), for 0 < t < tc
Depuration period: CF (t) / CW = (k1/k2)*(e-k2(t- tc)-e-k2t) for t > tc
Where,
CF (t): Concentration in fish as a function of time (µg test substance/kg wet weight)
Cw : Concentration in water during uptake period (µg test substance/L)
t: Time from start of exposure (day)
tc: Time at the end of the uptake phase (day), in this study day 44
k1: Uptake rate constant (day -1)
k2: Depuration rate constant (day -1)
Both formulas were combined to fit the measured data using the individual fish and water values at each measurement time with the SAS-Procedure NLIN (see appendix C).
The time to reach 50% depuration (or depuration half-life, t1/2) is calculated according to the formula: t1/2 = ln 0.5 / -k2. Similarly the time to reach 95% of the steady state concentration during uptake was calculated as: ln 0.05 / -k2.

Growth correction and lipid normalization
Since growth leads to a “dilution” of the test substance in the fish, the BCF calculation should be corrected for the growth rate. The depuration rate constant k2 consists of the true depuration rate constant and the growth rate constant. For the evaluation of the growth rate constants the individual weight data were converted to natural logarithms and plotted vs. time (day) separately for each test group, including data from the start of the uptake. A linear least squares correlation was calculated for each group and the variances of the slopes (growth rates) were statistically evaluated using student t-test (or ANOVA). Since there was a statistically significant difference between the slopes, the test and control data were not pooled and a separate fish growth rate constant for the study for each test group (kg(control) and kg(dose)) was calculated (see appendix C). The calculated growth rate constant can be subtracted from the depuration rate constant (k2) to give the growth-corrected depuration rate constant (k2g) and the BCFK is corrected for growth according to the equation:

BCFkg = k1 / (k2-kg)

BCF results should be adjusted to a guideline standard fish lipid content of 5% (w/w). A lipid normalization factor (Ln) represents the mean wet weight lipid fraction in fish (control fish at each sampling point). The BCF values (BCFSS, BCFK, and BCFKg) were lipid normalized as follows (using BCFK as an example):

BCF kl = (0.05/Ln) * BCFk

VALIDITY CRITERIA
• Temperature variation is less than ±2°C.
o In this study, the instantaneous test temperature was generally 12-13°C in all aquaria throughout the test period.
• Oxygen concentration ≥60% of the saturation value
o In this study, the measured dissolved oxygen was never less than 60% of the air saturation value in any aquarium throughout the test period.
• Test concentrations within ±20% of nominal or mean of determined concentrations during the uptake phase.
o In this study, test concentrations remained within ±20% of the mean of determined concentrations during the 44-day uptake phase. Only on day 42 of the uptake period the measured concentration (124.2%) was slightly higher than 20% of the mean of determined concentration but had no considerable influence on the overall mean measured results.
• ≤10% mortality in the control and treatment groups
o In this study, there was 0.95% mortality in the treatment group (1 fish out of 105).

All the validity criteria were achieved, and this study is considered valid.

Lipid content:

2.2 %

Time point:

other: uptake period, day 3

Lipid content:

3 %

Time point:

other: uptake period, day 44

Lipid content:

3.1 %

Time point:

other: depuration period, day 107

Conc. / dose:

0.2 µg/L

Temp.:

13 °C

pH:

8

Type:

BCF

Value:

6 356 L/kg

Basis:

whole body w.w.

Time of plateau:

28 d

Calculation basis:

steady state

Remarks on result:

other: normalized to 5% lipid content

Key result

Conc. / dose:

0.2 µg/L

Temp.:

13 °C

pH:

8

Type:

BCF

Value:

7 093 L/kg

Basis:

whole body w.w.

Time of plateau:

28 d

Calculation basis:

kinetic, corrected for growth

Remarks on result:

other: growth corrected and lipid normalized to 5% lipid content

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

4.6 d

Remarks on result:

other: depuration half-life

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

5.1 d

Remarks on result:

other: growth-corrected hal-life

Rate constant:

growth rate constant (d-1)

Value:

0.015

Rate constant:

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

Value:

537

Rate constant:

overall depuration rate constant (d-1)

Value:

0.15

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

0.135

Details on kinetic parameters:

The measured data from the concentration group from whole fish as well as edible and non-edible portions fit well to a first order kinetic model allowing an estimation of the uptake and depuration rate constants based on simultaneous curve fitting. The data illustrate that steady state was reached during the uptake period, after the sampling on day 44. After the start of depuration, the concentrations in fish progressively declined. After 63 days in clean water the whole-body residues in fish from the concentration group had declined to 4% of the mean steady state concentration (CFss).
The non-edible portion was analyzed separately from the remaining fish (head and carcass including bones, organs, skin and fins). Since bioconcentration should reflect the partitioning of the substance from water into the tissues of the fish, sorption to undigested food in the gut lumen would lead to erroneously high BCF estimates when based on the total fish according to guideline recommendations.


Overall the measured BCFss values were very similar to the calculated BCFK values indicating that steady state was reached, and that uptake and depuration follow first order kinetics. The most relevant BCF is the BCFK normalized to 5% lipid content (BCFKL) because it incorporates all measurements during uptake and depuration and since it removes the influence of the test fish lipid content. Since the BCFss and BCFK values were nearly identical, the overall lipid value was used to normalize all BCF values.

Details on results:

WATER QUALITY PARAMETERS
The pH was stable during the whole test period and was in a range of 8.0 – 8.2. The concentrations of dissolved oxygen were maintained in a range between 8.3 – 10.3 mg/L in the test vessels and were not less than 60% of the maximum saturation at the test temperature of 13 °C during the test. The instantaneous test temperature was generally 13 ±1°C in all aquaria throughout the test period. TOC values determined in the control group ranged from 7.8 - 10.5 mg/L during the test period due to the use of acetone as solvent. The TOC values determined in the used water before addition of acetone in the solvent control ranged from 0.6 – 1.4 mg/L. The measured value for hardness in the mixed water supply was at 1.02 mmol/L (approx. 102 mg/L CaCO3) at study day 0.
In conclusion, measurements of pH, dissolved oxygen and temperature were comparable between the control and treatment group and did not appear to be influenced by test substance concentration. Measurements of TOC and hardness were in the expected range. The guideline requirements for water quality parameters were satisfied.

MORTALITY AND SIGNS OF TOXICITY
No mortalities or signs of toxicity were observed in the control group and one mortality was observed in the treatment group over the whole test period.

FISH GROWTH
The growth data for fish at the start of the experiment and during the test are presented in Tables 11 and 12 respectively. The natural logarithm of fish weight vs. time (day) is plotted separately for each test group in Figure 1 and the slopes of the regression lines were taken as the growth rates. Both wet weight and total length were measured; however, growth rates were calculated based only on wet weight since this is the most relevant metric to evaluate growth.
The wet weight of all fish used in the experiment was determined on day 0 and ranged from 0.7 – 1.0 g/fish with a mean value of 0.85 g. During the test growth rates of 0.0087 and 0.0137/day were calculated for the control and 0.2 µg/L test group, respectively and were statistically evaluated to assess for differences among the test groups. Since there was a statistically significant difference between the slopes, the test and control data were not pooled and a separate fish growth rate constant for the study for each test group (kg(control) and kg(dose)) was calculated and used for “growth-corrected” calculations. Since juvenile fish were used, growth was expected during the experiment.

Measured concentration in test solution and fish tissue
Overall, in the concentration group 0.2 µg/L the concentration of 14C-2-(5-chloro(2H)-benzotriazole-2-yl)-4-(methyl)-6-(tert-butyl)phenol in the whole fish on day 14 was within 20% of the mean measured concentration in fish during the uptake period, indicating that the uptake had become approximately asymptotic with respect to time (reached plateau). The kinetic calculations indicate that 95% of the steady state concentration was reached after 44 days. Since the concentration in fish decreased to 80% of the mean measured concentration in fish on day 21, consequently, the measured fish values from day 28 till day 42 were considered for the calculation of the steady state bioconcentration factor (BCFss) and varied between 89 – 96% of the mean measured concentration in fish. In addition, the last day of uptake (day 44) was unpredictably slightly higher than >20% of the mean measured concentration in fish (123%) but was included as worst-case scenario concentration into the calculation of the steady state bioconcentration factor (BCFss). The BCFss was 3559 ± 561 L/kg, determined as mean value of CF(t)/CW from days 28 - 44. When normalized to a 5% lipid content in fish (using the mean lipid), the steady state bioconcentration factor (BCFssL) was 6356 L/kg.
The BCFss was determined as mean value of CF(t)/CW from days 28 - 44.
The BCFss was 3559 ± 561 L/kg and based on centrifuged test solutions the BCFss was 7136 ± 1124 L/kg. When normalized to a 5% lipid content in fish (using the mean lipid), the steady state bioconcentration factor (BCFssL) was 6356 L/kg and for centrifuged test solution the BCFssL was at 12743 L/kg.
The results of centrifuged samples were below 500 dpm/ 10 mL and should therefore be taken with care, since they are not fully reliable.

Uptake and depuration rate constants and bioconcentration factors (BCF) for the whole fish based on measured and calculated data.
Parameter	0.2 µg/L
kg(growth rate constant; day^-1) (standard error)	0.0148 (0.0004)
k1, (overall uptake rate constant, L/kg/day) (95% confidence interval)	537 (445-629)
k2, (overall depuration rate constant, day^-1) (95% confidence interval)	0.15 (0.13-0.18)
k2g(growth-corrected depuration rate constant, day^-1):	0.1352
CFss, (concentration in fish at steady-state, µg/kg) (mean (days 3-44) ±standard deviation)	562±88.5
CW(concentration in the water, µg/L) (mean (days 0 – 44) ±standard deviation)	0.158±0.022
Ln(lipid normalization factor) (end of uptake)	0.028
BCFss (steady-state BCF; L/kg) (mean (days 3-44) ±standard deviation)	3559±561
BCFssL(lipid normalized steady-state BCF; L/kg)	6356
BCFK(kinetic BCF; L/kg)	3580
BCFKg(growth-corrected kinetic BCF; L/kg):	3972
BCFKL(lipid-normalized kineticBCFKg; L/kg)	6393
Geometric mean BCFKLg^[a], L/kg	7093
t1/2, (depuration half-life; day)	4.6
t1/2g(growth-corrected half-life, day):	5.1
Time to 95% steady state (growth-corrected, day):	20
[a] The most relevant BCF in this study is the geometric mean growth corrected kinetic BCF normalized to 5% lipid content, BCFKLG.

Uptake and depuration rate constants and bioconcentration factors (BCF) for the whole fish based on measured and calculated data (based on centrifuged test solutions).
Parameter	0.2 µg/L
kg(growth rate constant; day^-1) (standard error)	0.0148 (0.0004)
k1, (overall uptake rate constant, L/kg/day) (95% confidence interval)	1077 (892-1261)
k2, (overall depuration rate constant, day^-1) (95% confidence interval)	0.15 (0.13-0.18)
k2g(growth-corrected depuration rate constant, day^-1):	0.1352
CFss, (concentration in fish at steady-state, µg/kg) (mean (days 3-44) ±standard deviation)	562±88.5
CW(concentration in the water, µg/L) (mean (days 0 – 44) ±standard deviation)	0.079±0.008
Ln(lipid normalization factor) (end of uptake)	0.028
BCFss (steady-state BCF; L/kg) (mean (days 3-44) ±standard deviation)	7136±1124
BCFssL(lipid normalized steady-state BCF; L/kg)	12743
BCFK(kinetic BCF; L/kg)	7180
BCFKg(growth-corrected kinetic BCF; L/kg):	7966
BCFKL(lipid-normalized kineticBCFKg; L/kg)	12821
Geometric mean BCFKLg^[a], L/kg	14225
t1/2, (depuration half-life; day)	4.6
t1/2g(growth-corrected half-life, day):	5.1
Time to 95% steady state (growth-corrected, day):	20
[a] The most relevant BCF in this study is the geometric mean growth corrected kinetic BCF normalized to 5% lipid content, BCFKLG.

The results of centrifuged samples were below 500 dpm/ 10 mL and should therefore be taken with care, since they are not fully reliable as the scintillator needs a minimum activity of 500 dpm/ 10 mL for calculation of reliable results, comparable to a limit of quantification (LOQ).

Validity criteria fulfilled:

yes

Conclusions:

This study assessed the bioconcentration potential of 14C-2-(5-chloro(2H)-benzotriazole-2-yl)-4-(methyl)-6-(tert-butyl)phenol in rainbow trout (Oncorhynchus mykiss). The test was conducted based on the guidelines US EPA OCSPP 850.1730 and OECD 305.
The fish were exposed to one solvent control group and one concentration group of test substance at 0.2 µg/L in a flow-through-system for an uptake period of 44 days followed by a depuration period in clean water of 63 days.
Over the entire test all water quality parameters were maintained within acceptable limits. No toxic effects (i.e. mortality) or changes in behavior or appearance were observed in the test treatment organisms in comparison to the control group. Since there was a statistically significant difference between the slopes of the growth rate, the test and control data were not pooled and a separate fish growth rate constant for the study for each test group (kg(control) and kg(dose)) was calculated and used for “growth-corrected” calculations. Since juvenile fish were used, growth was expected during the experiment. The lipid content of control fish sampled over the test period remained constant considering the variability of individual values and the overall mean lipid content from the exposure period (2.8%) was used for lipid normalization calculations.
The theoretical concentration and homogeneity can be confirmed by the measurement of radioactivity from 3 different regions of the test solution tank centrifuged and uncentrifuged samples prior to the start of exposure (measurements without a GLP status). Since there was a difference in the results in centrifuged and uncentrifuged samples, additionally to concentration measurements of uncentrifuged samples also concentrations of centrifuged samples were measured as worst-case scenario and were used in subsequent analyses.
Test substance concentrations in test solution were determined on 9 occasions during uptake by measuring the total radioactivity. The mean-measured concentrations of the test substance during the uptake period in test solution were 0.158 ± 0.022 µg/L. The concentration was kept constant within the range of ±20% of the mean measured concentration throughout the exposure period. Only on day 42 of the uptake period the measured concentration (123.4%) was slightly higher than 20% of the mean of determined concentration but had no considerable influence on the overall mean measured results. Between day 0 and day 44 of the uptake period the measured concentrations of the centrifuged samples varied between 84 and 123% of the mean measured concentration throughout the uptake period.
For centrifuged test solutions only on day 29 of the uptake period the measured concentration in centrifuged test solutions (122.6%) was slightly higher than 20% of the mean of determined concentration but had no considerable influence on the overall mean measured results. The mean concentrations of 14C-2-(5-chloro(2H)-benzotriazole-2-yl)-4-(methyl)-6-(tert-butyl)phenol in centrifuged test solution during the uptake phase (through day 44) were 0.079 ±0.008 µg/L (100% of the mean determined concentration). Between day 0 and day 44 of the uptake period the measured concentrations of the centrifuged samples varied between 90 and 123% of the mean measured concentration throughout the uptake period.
During depuration the concentration in test solution was measured on 3 occasions and in fish on 10 occasions.
Test substance concentrations in fish were determined on 9 occasions during uptake by measuring the total radioactivity. Total radioactive residues in fish were measured separately in edible (e.g. fillet) and non-edible (e.g. remaining carcass) portions and the whole fish value was calculated from the weight normalized sum of the individually measured portions. The concentration in fish reached 95% steady state within 44 days based on the kinetic calculations.
Based on uncentrifuged test solutions, overall the measured steady state bioconcentration factor (BCFss) value (3559 ±561) was very similar to the calculated kinetic (BCFK) value (3580) indicating that steady state was reached, and that uptake and depuration follow first order kinetics. The accumulation in the edible fish portions was less than in the non-edible portions. However, the most relevant BCF is the growth corrected kinetic BCF normalized to 5% lipid content (BCFKLg) for the whole fish because it incorporates all measurements during uptake and depuration and since it removes the influence of the test fish lipid content.
In conclusion, based on uncentrifuged test solutions, the bioconcentration factor BCFKLg was 7093 for the whole fish based on total radioactive residues of the test substance.

Based on centrifuged test solutions the BCFss was 7136 ± 1124 L/kg. When normalized to a 5% lipid content in fish (using the mean lipid), the steady state bioconcentration factor (BCFssL) was 12743 L/kg for centrifuged test solution and the bioconcentration factor BCFKLg was 14225 for the whole fish based on total radioactive residues of the test substance.
The results of centrifuged samples were below 500 dpm/ 10 mL and should therefore be taken with care, since they are not fully reliable as the scintillator needs a minimum activity of 500 dpm/ 10 mL for calculation of reliable results, comparable to a limit of quantification (LOQ).
Throughout the test, the concentration of total organic carbon in the test vessels did not exceed the concentration of organic carbon originating from the test substance and solvent by more than 10 mg/L (± 20%), as recommended in the test guideline. Since organic matter content can have a significant effect on the amount of freely dissolved test substance during flow-through fish tests, especially for highly lipophilic substances uneaten food and feces were siphoned daily from the aquaria shortly after feeding and aquaria were kept as clean as possible throughout the test to keep the concentration of organic matter as low as possible.

The results in this study are consistent with all validity criteria and the test is valid according to the guidelines of this study. No deviations from test guidelines or other incidents occurred during the course of the reported test which may have influenced the results.

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1997-09-16 to 1998-02-04

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study and in accordance to OECD guideline. No depuration phase.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Bioaccumulation study of chemical substance in fish body; Testing methods relating New Chemical Substances (Kanpogyo No. 5, Yakuhatsu No. 615, 49 Kikyoku No. 392, July 13, 1974

Deviations:

no

GLP compliance:

yes

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms: Test material in the test fish was analyzed 2, 4, 6, and 8 weeks four times in total for Level 1 and 2 areas and for Level 3 area was analyzes 2, 4, 6, 8 and 10 weeks, 5 times in total, after the initiation of exposure, using two fish samples each time. The analysis of test material in the control fish was performed before the start and after the completion of exposure, using two fish samples each time.
- Sampling intervals/frequency for test medium samples: The test material in the test water was analyzed twice weekly, 16 times in total, for both Level areas 1 and 2 and Level area 3 was analyzed 20 times in total during the exposure period, using one sample each time

Vehicle:

yes

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

PREPARATION AND APPLICATION OF TEST SOLUTION
Preparation of stock solution
(1) Dispersant: HCO-20
(2) Preparation method: The test material and 40 times amount of HCO-20 were dissolved in acetone, the acetone was distilled away, and obtained a 1000mg/L stock solution dissolving in ion exchange water.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Carp (Cyprinus carpio)
- Source: Sugishima Fish Farm (Address: 123-2, Gun-chiku Ichiban-cho, Yatsushiro City, Kumamoto 866)
- Length at study initiation (lenght definition, mean, range and SD): Level area 1 & 2: mean = 9.2 cm; Level area 3: mean = 9.8 cm
- Weight at study initiation (mean and range, SD): Level area 1 & 2: mean = 20.5 g; Level area 3: mean = 23.6 g
- Health status: The fish were macroscopically observed on receipt and those demonstrating any abnormality removed. Then the fish were cultivated for one day under flow through conditions following an external disinfection in the receiving tank.
- Description of housing/holding area: aquarium
- Feeding during test
- Food type: Formula pellet feed for carp (Nippon formula Feed Mfg. Co. Ltd)
- Amount: Corresponding to approximately 2 % of the test fish weight fed each day.
- Frequency: Twice per day

ACCLIMATION
- Acclimation period: Fish were acclimated in an aquarium. During acclimatization, fish demonstrating any abnormality were eliminated. Then, the fish were cultivated under flow through conditions at 25+/-2 °C for 48 hours. Finally they were transferred to a test aquarium, dipped and cultivated for 27 days at the same temperature under flow through conditions.
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: Formula pellet feed for carp (Nippon formula Feed Mfg. Co. Ltd); Corresponding to approximately 2 % of the test fish weight fed each day.
- Feeding frequency: Twice per day
- Health during acclimation (any mortality observed): No data - any fish demonstrating mortality were removed.

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

8 - 10 wk

Test temperature:

25+/-2 °C

pH:

at initiation of exposure: 8.1
at termination of exposure: 7.8

Dissolved oxygen:

Level 1 area: 6.9-7.8 mg/L
Level 2 area: 7.5-8.1 mg/L
Level 1 and 2 area controls: 7.1-7.6 mg/L
Level 3 area: 7.4-8.0 mg/L
Level 3 area control: 7.7-8.1 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): no data
- Material, size, headspace, fill volume: 100 L glass aquarium
- Type of flow-through (e.g. peristaltic or proportional diluter): no data
- Renewal rate of test solution (frequency/flow rate): The stock solution and the dilution water were supplied to a test tank to a total of 1155 L/day, at rates of 2 mL/min and 800 mL/min, respectively.
- No. of organisms per vessel: Level area 1 & 2: 11; level area 3: 18; controls: 5

TEST MEDIUM / WATER PARAMETERS
- in accordance to the guideline

TEST MATERIAL CONCENTRATIONS
Based on the preliminary test results for 48-hour LC50 and the analytical sensitivity of test material, the test material concentrations were determined as follows:
Level 1 area 0.5 mg/L
Level 2 area 0.05 mg/L
Level 2 area 0.005 mg/L
A control area was also established for a blank test.

Nominal and measured concentrations:

Nominal:
Level 1 area 0.5 mg/L
Level 2 area 0.05 mg/L
Level 3 area 0.005 mg/L

Reference substance (positive control):

no

Lipid content:

ca. 3.8 %

Time point:

start of exposure

Remarks on result:

other: Level area 1 & 2

Lipid content:

ca. 3.7 %

Time point:

start of exposure

Remarks on result:

other: Level area 3

Type:

BCF

Value:

54 - 109

Basis:

whole body w.w.

Remarks on result:

other: Level area 1

Remarks:

Conc.in environment / dose:0.5 mg/L

Type:

BCF

Value:

196 - 802

Basis:

whole body w.w.

Remarks on result:

other: Level area 2

Remarks:

Conc.in environment / dose:0.05 mg/L

Type:

BCF

Value:

548 - 895

Basis:

whole body w.w.

Remarks on result:

other: Level area 3

Remarks:

Conc.in environment / dose:0.005 mg/L

Metabolites:

An extremely small metabolic peak was observed ahead of the test material peak on reverse phase HPLC chromatographic analysis of Level area 1 and 2 fish. According to 3-D chromatography, the UV absorption spectrum pattern of this metabolic peak was the same as the test material. The atomic mass number was confirmed to be 226 and 313 by HPLC-MS. Based on the above, the metabolite was assumed to have a similar basic structure to the test material, but it was not identified due to the extremely small amount. The BCF obtained by converting the metabolic peak to test material was 1.9-5.5 in level 1 area and 14-90 in level 2 area. The metabolic peak was not observed in level 3 area.

Description of key information

Based on the results of an OECD 305 study, the substance is highly bioaccumulative in aquatic organisms.

Additional information

For the assessment of the bioaccumulative potential of CAS 3896-11-5 two experimental studies are available. The key study was conducted based on the guidelines US EPA OCSPP 850.1730 and OECD 305. Oncorhynchus mykisswere exposed to one solvent control group and one concentration group of test substance at 0.2 µg/L in a flow-through-system for an uptake period of 44daysfollowed by a depuration period in clean water of 63 days.The test concentration of 0.2µg/L was below the limit of solubility of the test compound in water (4 µg/L).Based on uncentrifuged test solutions, overall the measured steady state bioconcentration factor (BCFss) value (3559 ±561) was very similar to the calculated kinetic (BCF_K) value (3580) indicating that steady state was reached, and that uptake and depuration follow first order kinetics. The accumulation in the edible fish portions was less than in the non-edible portions. However,the most relevant BCF is the growth corrected kinetic BCF normalized to 5% lipid content(BCF_KLg) for the whole fish because it incorporates all measurements during uptake and depuration and since it removes the influence of the test fish lipid content.

In conclusion, based on uncentrifuged test solutions, the bioconcentration factor BCF_KLgwas 7093 for the whole fish based on total radioactive residues of the test substance.

The supporting study was conducted at the Chemicals Inspection & Testing Institute, Japan, The Chemical Biotesting Center, Kurume Laboratory from 1997 to 1998. Japanese rice fish (Oryzias latipes) were exposed to three concentrations of the test compound (0.5, 0.05, and 0.005 mg/L) in a continuous flow-through test design for 8 weeks (concentration 0.5 and 0.05 mg/L) and 10 weeks (concentration 0.005 mg/L) without a subsequent depuration phase. The stock solutions were prepared with 40-fold HCO-20 as solubilizer. Two fish from each concentration group were sampled at weeks 2, 4, 6, 8 (and 10 for the 3rd concentration group) and analyzed for the test compound. The resulting BCF values ranged from 54-109 L/kg, 196-802 L/kg, and 548-895 L/kg for the test groups 0.5, 0.05, and 0.005 mg/L, respectively. This study underestimated the true BCF probably due to the high test substance concentrations.