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Bioaccumulation: aquatic / sediment

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Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2019-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test
Radiolabelling:
yes
Details on sampling:
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). On day 21 the gastro-intestinal tracts of the 5 fish were combusted separately for a possible localization of the radioactivity. The result will be used for discussion.
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 and depuration period:
Sampling time Test solution samples / concentration No. of fish to sample / concentrationa
Uptake period
Day 0 2 -
Day 1 2 5
Day 2 2 5
Day 3 2 5
Day 7 2 5
Day 14 2a 5
Day 21 2 5
Day 28 2 5
Day 31 2 5
Day 35 2 5

Depuration periodb
D.-Day 35.125 2 5
D.-Day 36 2 5
D.-Day 38 2 5
D.-Day 42 2 5
D.-Day 49 - 5
a = from the control group, three fish are used for lipid analyses and two for background correction
- = no samples taken
b = during the depuration period test solution samples were taken until 2 consecutive measurements were less than 1% of nominal.

At the end of the uptake period, the bioconcentration factor at steady state was >10 therefore a depuration period was needed according to the test guideline. The remaining fish were transferred to aquaria with test water without test substance. These aquaria received test water at the same flow rate as during the uptake period. The depuration period was 14 days.
Sampling time schedule during the additional uptake period 1 to 5 hours
(test group 0a and 1a):
Sampling time Test solution samples / concentration No. of fish to sample / concentrationa
Uptake period
1 h 2 5
3 h 2 5
5 h 2 5
a = from the control group, only two fish were inserted and used for background correction
Vehicle:
no
Details on preparation of test solutions, spiked fish food or sediment:
A stock solution (50 µg/L) was prepared using a solid-liquid (slow-stir) saturator technique. Briefly, this technique involves coating the bottom surface of a stainless-steel stock solution tank with the test substance to generate a saturated aqueous solution. The [14C] test substance 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 acetonitrile was completely evaporated, afterwards the test substance was re-dissolved in approx. 20 mL acetone. The acetone solution was checked for complete dissolution of the test substance, then the solution was distributed on the bottom of the stock solution tank. All the acetone was then completely evaporated to leave a thin layer of test substance coating the bottom of the stock solution tank. Afterwards 50 L of test water was added (taking care to minimize the disturbance of the test substance layer) and the stock solution was stirred with a paddle stirrer at the test temperature for at least 3 hours or overnight. Since all acetone was evaporated prior to adding water, no solvent was present in the final test solution and no solvent control was required.
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 (measurements without a GLP status). Since no difference in the radioactivity content was observed between centrifuged and uncentrifuged samples during the concentration verification and homogeneity verification, further analyses and results were based on uncentrifuged water samples.

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.

Theoretical radioactivity in the solutions:
3570 dpm / 10 mL in the test solution, 0.5 µg/L
35700 dpm / 1 mL in the stock solution, 50 µg/L.

The in-life exposure was conducted in a continuous flow through system (see Figure 5). 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 5 days before insertion of the fish to allow equilibration.
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 GbR, Dorfstrasse 7, 98646 Trostadt, 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 0.5 g/fish (±0.06, SD) with a range of 0.4 – 0.6 g/fish. The minimum fish body weight was 90.5% of the maximum body weight (see Table 11 in Appendix).
Body length (overall mean): 3.9 cm ±0.9
Hatch date: 31 May 2019
Arrival in the test facility: 02 Jul 2019
The fish were approximately 1 month old at the start of exposure.
Reason for species selection: Rainbow trout is used routinely for toxicity tests in the testing facility and was requested by the authorities.


HOUSING AND DIET
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) and live juvenile brine shrimp (Artemia nauplii) 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:
<5%
Route of exposure:
aqueous
Justification for method:
aqueous exposure method used for following reason: for comparison to the regulatory BCF threshold
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
35 d
Total depuration duration:
14 d
Hardness:
The measured values for hardness in the control and in the treatment group were 1.04 mmol/L (approx. 104 mg/L CaCO3) at study day 0
Test temperature:
13 °C
pH:
8.0 – 8.4
Dissolved oxygen:
9.0 – 10.2 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.
TOC:
0.7 – 1.2 mg/L during the test period
Salinity:
n/a
Conductivity:
270 µS/cm
Details on test conditions:
Exposure: Flow through system
Water temperature: 13°C
Aeration: The test water is saturated with oxygen. An aeration in the test vessels is used only if otherwise the maintenance of a sufficient oxygen concentration of >60% of the saturation concentration in the test vessels cannot be ensured.
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 70 fish with a mean body weight of 0.5 g (wet weight) were inserted into each test vessel. Therefore, based on the flow rate of 504 L/d the loading was 0.07 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.
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.

Each test group consisted of 1 aquarium with 70 fish randomly inserted at start of test exposure. Each test group aquaria were identified with appropriate labels.
An additional glass aquarium (test group 1a) (approx. 24 L) was set up next to the 100 L aquarium from test group 1 and supplied with the same test solution from the 100 L aquarium.
The control group (0a) was a test water control only and fish were taken from the housing tanks. As sampling time was at 1, 3 and 5 hours after start of exposure the 6 randomly distributed fish for test group 0a were kept in the housing tank separated from the other fish of the housing tank via a net.
The same batch of fish was used as well as the same weight range as at the start of the study (day 0). This additional aquarium was set up to show that uptake of the test substance is quick.

The control group was a test water control only. A solvent control group was not needed, since no solvent was present in the final test solution.

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: 0.5 µg/L
mean-measured concentrations of the test substance during the uptake period in test solution were 0.477 ± 0.036 µg/L
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 35
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 no statistically significant difference between the slopes, the test and control data were pooled and an overall fish growth rate constant for the study (kg) was calculated as the overall slope of the linear correlation (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):

BCFkl = (0.05 / Ln) * BCFk

VALIDITY CRITERIA
• Temperature variation is less than ±2°C.
o In this study, the instantaneous test temperature was generally 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 analytically determined concentrations during the uptake phase.
o In this study, test concentrations remained within ±20% of the mean measured concentration during the 35-day uptake phase.
• ≤10% mortality in the control and treatment groups
o In this study, there was one mortality in the test group 0.5 µg/L (1 out of 70 fish = 1.4%).

All the validity criteria were achieved, and this study is considered valid.
Lipid content:
1.77 %
Time point:
other: uptake period, day 1
Lipid content:
4.07 %
Time point:
other: uptake period, day 35
Lipid content:
3.8 %
Time point:
other: depuration period, day 49
Key result
Conc. / dose:
0.477 µg/L
Temp.:
13 °C
pH:
8
Type:
BCF
Value:
1 456 L/kg
Basis:
whole body w.w.
Time of plateau:
2 d
Calculation basis:
kinetic, corrected for growth
Remarks on result:
other: BCFklg, growth-corrected, lipid-normalized BCF
Conc. / dose:
0.477 µg/L
Temp.:
13 °C
pH:
8
Type:
BCF
Value:
1 623 L/kg
Basis:
whole body w.w.
Time of plateau:
2 d
Calculation basis:
steady state
Remarks on result:
other: BCFss, lipid-normalized
Elimination:
yes
Parameter:
DT50
Depuration time (DT):
0.5 d
Elimination:
yes
Parameter:
DT50
Depuration time (DT):
0.6 d
Rate constant:
growth rate constant (d-1)
Value:
0.018
Rate constant:
overall uptake rate constant (L kg-1 d-1)
Value:
1 166
Rate constant:
overall depuration rate constant (d-1)
Value:
1.27
Rate constant:
growth-corrected depuration rate constant (d-1)
Value:
1.252
Details on kinetic parameters:
The measured data from the concentration group from whole fish 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.
For the concentrations in fish (total and non-edible) the results for the two compartment model converged to the one compartment model.
For the concentration in edible fish no convergence was reached for the two compartment model. Although no convergence was reached, the edible concentrations in fish are still higher than the estimated values. Therefore, the two compartment model was fitted to the data of the depuration phase with and without day 35.
A better fit could only be reached without day 35.


The slopes of the two compartment model using only data of the depuration phase excluding day 35 may give a hint for the depuration.

alpha (Slope of Compartment A ) = 0.6547
beta (Slope of Compartment B) = 0.0227

These estimations should be taken with care, because no data during the uptake phase was considered and the depuration from day 35 to 35.125 was not considered, which is higher than the estimated slope alpha. As the concentration in the edible portion stays extremely low, an accumulation in the edible parts of the fish are negligible.

The data illustrate that steady state was quickly reached during the uptake period, after the sampling on day 2. After the start of depuration, the concentrations in fish progressively declined. After 7 days in clean water the whole-body residues in fish from both concentration groups had declined to 2% of the mean steady state concentration (CFss).
Based on the kinetic model, the calculated uptake rate constant (k1) was 1166 day-1 from the exposure concentration. The calculated depuration rate constant (k2) was 1.27 day-1 from the exposure concentration.
For statistics, the measurements from 1 to 5 hours were also included and it could be shown that the uptake of test substance was quick. However, these measurements were not included in the overall BCF calculations as they would not have changed the result.

The non-edible portion was analyzed separately as the head and the remaining fish (including: bones, gastro-intestinal tracts, internal organs, skin and fins). On day 21 the gastro-intestinal tracts of the 5 fish were combusted separately for a possible localization of the radioactivity. Radioactivity was mostly found in the gastro-intestinal tracts. Fish regularly drink water, so this high concentration could result from the test substance binding to food in the lumen of the gastrointestinal tract. As the lipid content of the fish diet is 16%, it is an attractive matrix to sorb hydrophobic chemicals. 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. However, the quick reduction of the test substance in whole fish indicates that the substance is quickly excreted.
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 only slightly different, the overall mean lipid value was used to normalize all BCF values.
Details on results:
This study assessed the bioconcentration potential of 14C-2-(2H-Benzotriazol-2’-yl)-4-methyl-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 control group and one concentration group of test substance at 0.5 µg/L in a flow-through-system for an uptake period of 35 days followed by a depuration period in clean water of 14 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. There was no statistically significant difference in fish growth rate between control and treatment groups during the experiment, therefore data from all groups were combined to determine the overall growth rate (kg) for “growth-corrected” calculations. 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 (3.2%) was used for lipid normalization calculations.
Test substance concentrations in test solution were determined on 10 occasions during uptake by measuring the total radioactivity. The mean-measured concentrations of the test substance during the uptake period in test solution were 0.477 ± 0.036 µg/L. The concentration was kept constant within the range of ±20% of the nominal concentration throughout the exposure period. During depuration the concentration in test solution was measured on 4 occasions and in fish on 5 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.
With a setup of an additional aquarium and sampling times after 1, 3 and 5 hours of exposure, it could be shown that uptake of the substance is very quick and steady state is reached within 2.4 days. In addition, on day 21 the gastro-intestinal tracts of 5 fish were combusted separately for a possible localization of the radioactivity. Radioactivity was mostly found in the gastro-intestinal tracts. Fish regularly drink water, so this high concentration could result from the test substance binding to food in the lumen of the gastrointestinal tract. As the lipid content of the fish diet is 16%, it is an attractive matrix to sorb hydrophobic chemicals. 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.
However, the quick reduction of the test substance in whole fish indicates that the substance is quickly excreted.
The concentration in fish reached 95% steady state within 2.4 days based on the kinetic calculations.
Overall the measured steady state bioconcentration factor (BCFss) values (1039) was quite similar to the calculated kinetic (BCFK) value (918) indicating that steady state was reached, and that uptake and depuration follow first order kinetics. The accumulation in the edible fish portions was very low and negligible. 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 the bioconcentration factor BCFKLg was 1456 for the whole fish based on total radioactive residues of the substance.
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.
Validity criteria fulfilled:
yes
Conclusions:
• Temperature variation is less than ±2°C.
o In this study, the instantaneous test temperature was generally 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 analytically determined concentrations during the uptake phase.
o In this study, test concentrations remained within ±20% of the mean measured concentration during the 35-day uptake phase.
• ≤10% mortality in the control and treatment groups
o In this study, there was one mortality in the test group 0.5 µg/L (1 out of 70 fish = 1.4%).

All the validity criteria were achieved, and this study is considered valid.
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 305 C (Bioaccumulation: Test for the Degree of Bioconcentration in Fish)
Qualifier:
according to
Guideline:
other: "Tests for chemical concentration in fish and shellfish" of the Environmental Preservation Operation No. 5, PAB Notification No. 615, and Notification No. 49-392 of the Basic Industries Bureau (dated July 13, 1974).
GLP compliance:
yes
Radiolabelling:
no
Details on sampling:
SAMPLING
From the test water tank:
- 5 mL of 1st Conc. Range (of 1 mg/L)
- 50 mL of 2nd Conc. Range (of 0.1 mg/L)
- 200 mL of 3rd Conc. Range (of 0.01 mg/L)

Using a single sample for one analysis, the test water samples from the 2nd and 3rd Conc. Range were analyzed twice a week, respectively, during the exposure period, totaling 16 analyses per Conc. Range. In addition, 2 test fish samples each from the 1st, 2nd, and 3rd Step Con. were analyzed 2, 4, 6, and 8 weeks after the initiation of exposure, totaling 4 analyses per Conc. Range. The test fish from the control Conc. Range was analyzed before initiation and after completion of exposure, using 2 fish per analysis.
Vehicle:
yes
Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION OF SPIKED WATER
- Dispersant: HCO-20 and olive oil
- Preparation of test solutions: The test substance, 20-fold HCO-20 (w/w), and 20-fold olive oil (w/w) were dissolved in acetone. After evaporation of acetone, the mixture was dissolved in ion-exchanged water to 400, 40, and 4 mg/L of the test substance for use as the stock solution for the test concentrations of 1.0, 0.1 and 0.01 mg/L, respectively.
- Control of 1st and 2nd conc. ranges: HCO-20 and olive oil were dissolved in acetone. After acetone was evaporated, the substance was dissolved in ion-exchanged water to prepare the respective stock solutions of HCO-20 and olive oil at a concentration of 8 g/L, respectively.
- Control of 3rd conc. range: HCO-20 or olive oil was dissolved in acetone. After acetone was evaporated, the substance was dissolved in ion-exchanged water to prepare the respective stock solutions of HCO-20 and olive oil at a concentration of 0.08 g/L, respectively.

Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: Carp
- Source: Sugishima fishery (Location: 123-2 Gunchiku Ichiban-cho, Yatsushiro, Kumakomo, Japan 866-0024)
- Length at study initiation: 1 mg/L and 0.1 mg/L test: 9.3 cm on average. 0.01 mg/L test: 9.9 cm on average.
- Weight at study initiation: 1 mg/L and 0.1 mg/L test: 21.9 g on average. 0.01 mg/L test: 26.3 g on average.
- Lipid content: 1 mg/L and 0.1 mg/L test: 3.6% on average. 0.01 mg/L test: 4.0% on average.
- Rearing condition: After macroscopically abnormal fish were eliminated on the receipt day, the test fish were kept in a drug bath, and then reared in running water for 1 day.
- Feeding during test: yes
- Food type: Formula pellets for carp (Nippon Formula Feed Manufacturing Co., Ltd.)
- Feeding method: food at ca. 2% equivalent to the body weight of test fish was divided into 2 for daily feeding. Feeding was stopped on the day before sampling the test fish.

ACCLIMATION
- Acclimation period: After rearing, the test fish were kept in a drug bath to remove parasites, transferred to an acclimatization water tank again for treatment with drugs, and acclimatized. Any abnormal fish were eliminated while acclimatizing, and the rest were reared in running water of 25 ± 2°C for 48 days. Then, fish were transferred to the test water tank, treated with drugs, and further reared in running water at the same temperature for 33 days (1st and 2nd concentration) or 22 days (3rd concentration).
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
8 wk
Test temperature:
25 ± 2°C
Dissolved oxygen:
6.8 to 7.8 mg/L in 1 mg/L test concentration ('1st Conc. Range')
7.0 to 7.8 mg/L in 0.1 mg/L test concentration ('2nd Conc. Range')
7.3 to 8.1 mg/L in 0.01 mg/L test concentration ('3rd Conc. Range')
7.2 to 8.0 mg/L in controls
Details on test conditions:
TEST SYSTEM
- Test vessel: 100-L glass water tank (1st and 2nd Conc. Ranges) and 70-L glass water tank (3rd Conc. Range)
- Flow-through conditions: The stock solution at a rate of 2 mL/min and test water at a rate of 800 mL/min were supplied to the test water tank at a rate of 1155 L/day.
- No. of organisms per concentration: 15 for 1 mg/L and 0.1 mg/L test groups (at initiation of exposure); 11 for 0.01 mg/L test group (at initiation of exposure).
- No. of organisms for the control: 5 animals per group (one control for the 1.0 and 0.1 mg/L test concentrations, and one control for the 0.01 mg/L test concentration).

TEST WATER
- Type: Ground water pumped in the area of Kurume Institute
- Water quality confirmation: The quality test or analysis results of water sampled on Aug. 1, 1997 was analysed once every 6 months). The quality test results or analytical values in each test item were below the concentration described in the “Water Quality Standards based on the Water Supply Law” (Health and Welfare Ministry Ordinance No. 69, revised Dec. 21, 1992), “Water Standards for Fishery” (Japan Fisheries Resources Conservation Association, dated March 1983), or the “OECD Guideline.

RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: Taken into consideration a preliminary 48-hour LC50 and the analytical sensitivity to the test substance, the test substance concentration was set.

OBSERVATION AND MEASUREMENTS
(1) Observation of test fish: Health condition and others of test fish were observed twice a day.
(2) Volume of test water: The test water volume was measured once a day and recorded using a graduated cylinder.
(3) Test water temperature: The temperature of the test water was measured once a day and recorded using an alcohol thermometer.
(4) Dissolved oxygen: The concentration of dissolved oxygen in test water was measured twice a week using a dissolved oxygen meter.
(5) Others: Carp waste and tank wall taint were cleaned once a day during the study period.
Nominal and measured concentrations:
Nominal concentrations: 1.0, 0.1 and 0.01 mg/L and a blank control.
Measured concentrations:
- Week 2: 0.806, 0.0685 and 0.00763 mg/L.
- Week 4: 0.810, 0.0670 and 0.00765 mg/L
- Week 6: 0.819, 0.0669 and 0.00776 mg/L
- Week 8: 0.834, 0.0686 and 0.00785 mg/L

Throughout the report the 1, 0.1 and 0.01 mg/L test concentrations are referred to as '1st Conc. Range', '2nd Conc. Range' and '3rd Conc. Range', respectively.
Reference substance (positive control):
no
Details on estimation of bioconcentration:
BASIS INFORMATION
- Test frequency: Using a single sample for one analysis, the test water samples from the 2nd and 3rd conc. range were analyzed twice a week, respectively, during the exposure period, totaling 16 analyses per conc. range. In addition, 2 test fish samples each from the 1st, 2nd, and 3rd Step con. were analyzed 2, 4, 6, and 8 weeks after the initiation of exposure, totaling 4 analyses per conc. range. The test fish from the control conc. range was analyzed before initiation and after completion of exposure, using 2 fish per analysis.

Lipid content:
3.6 %
Time point:
start of exposure
Remarks on result:
other: 1st and 2nd Concentration Area
Lipid content:
4 %
Time point:
start of exposure
Remarks on result:
other: 3rd Concentration Area
Type:
BCF
Value:
123 - 494
Remarks on result:
other: Conc.in environment / dose:1000 µg/L
Type:
BCF
Value:
130 - 295
Remarks on result:
other: Conc.in environment / dose:100 µg/L
Type:
BCF
Value:
44 - 220
Remarks on result:
other: Conc.in environment / dose:10 µg/L
Details on results:
- Observation of test fishs: No abnormality was found.
See any other information on results incl. tables for the test substance concentration in test water and bioconcentration factor.

Bioconcentration factor (BCF).

Test concentration (mg/L)

Week 2

Week 4

Week 6

Week 8

1

152

282

280

214

123

192

494

200

0.1

154

150

144

130

271

295

167

175

0.01

44

175

183

131

86

84

220

135

Description of key information

The BCF of the substance is clearly below 2000.

Key value for chemical safety assessment

Additional information

The key study (BASF, 35F0235/19E005a, 2020) assessed thebioconcentration potential of 14C-2-(2H-Benzotriazol-2’-yl)-4-methyl-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 control group and one concentration group of test substance at 0.5 µg/L in a flow-through-system for an uptake period of 35days followed by a depuration period in clean water of 14 days. Over the entire test all water quality parameters were maintained within acceptable limits. The bioconcentration factor BCFKLg was 1456 for the whole fish based on total radioactive residues of the test substance.

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.

Furthermore, a supporting study according to OECD TG 305C is available. This study was conducted at the Safety Center for Chemicals, Kurume Institute, Chemicals Inspection & Testing Inst., Japan from 1997 to 1998. Japanese rice fish (Oryzias latipes) were exposed to three concentrations of the test compound (1, 0.1, and 0.01 mg/L) in a semi-static test design for 8 weeks without a subsequent depuration phase. The stock solutions were prepared with 20-fold HCO-20 (w/w) and 20-fold olive oil (w/w) as solubilizer. Two fish from each concentration group were sampled at weeks 2, 4, 6, and 8 and analyzed for the test compound. The resulting BCF values ranged from 123-494 L/kg, 130-295 L/kg, and 44-220 L/kg for the test groups 1, 0.1, and 0.01 mg/L, respectively.