Bis(2-ethylhexyl) tetrabromophthalate

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Deviations: water temperature (see "Principle of method if other than guideline"

Principles of method if other than guideline:

Deviations:
• The validity criteria set by the OECD guideline 305 allow a deviation of the experimental temperature in water of 15 ± 2°C [4] during testing. The water temperature deviated at the beginning of the experiment from this range as specified in the study plan. During the first 3 days and at day 7 of exposure, the temperature fell about 0.7°C below 13°C. Nevertheless, both vessels (control and treatment) experienced the same temperature regimen. Similarly, both vessels experienced a short warming event on day 39, as the temperature rose above the limit of 17°C to 19°C due to a technical problem.
• Control samples of fish from the uptake phase contained test item at concentrations > LOQ. The relative proportion of the detected content was below 1 % of the mean concentration of the treated group.

GLP compliance:

yes (incl. QA statement)

Details on sampling:

Sampling:
- Repeated samplings were done for both fish populations, removing 6 individuals at each sampling event for chemical analyses
- During uptake phase, fish were sampled twice at days 14 and 28
- At each sampling date, the individual weight of the sampled fish was determined
- Each fish sample was homogenized using an ultra turrax and extracted with acetone.

Vehicle:

no

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

PREPARATION OF EXPERIMENTAL DIET:
For each of the experiments (pre- and main test) new batches of feed were prepared with test substance by solvent spiking. The commercial feed Inicio Plus (biomar, Denmark) with a pellet size of and 1.1 mm was used for experimentation. An amount of 153.19 mg of test substance was dissolved in 5 ml diethylether (taken into account the purity of 95.5 %, this corresponds to a concentration of 29.259 mg/mL TBPH spiking solution). A batch of 100 g fish feed was placed in a pear-shaped drying flask (2 L volume) connected to a rotary evaporator equipped with a stainless steel capillary solvent-inlet tube to obtain a simple vacuum spiking apparatus. 3.930 ml of the above mentioned spiking solution (115 mg TBPH) was then spread among feed particles through a spray application while mixing by rotation to ensure a homogenous distribution of test substance on pellets. During spiking, pressure of the vacuum system was set to approximately 700 mbar. The flask was kept in a water bath at approximately 40°C. After dosing of the test item, the capillary inlet tube was sealed with parafilm and the pressure was reduced to approximately 400 mbar for another 10-20 minutes of rotation to ensure complete evaporation of the solvent. The spiked pellets were dispersed on the bottom of a stainless-steel container and left for at least 24 h in the fume hood before application, in order to remove potential solvent residues in spiked feed. Control diets were prepared exactly the same way as the spiked feed, but without test substance in the spiking solvent.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

SPECIFICATION:
- Source: Fischzucht Störk, Wagenhausen 8, 88348 Bad Saulgau, Germany
- Holding: The fish were held in water of the same quality as used in the test (purified drinking water) until the start of exposure. Test fish were held for at least 14 days prior to the test under equivalent water quality, feeding and illumination conditions to those proposed for use in the test.
- Developmental state: The criteria of the test guideline [4] were followed. Juvenile fish of similar weight (2.064 ± 0.200 g) and length (5.566 ± 0.152 cm) were selected for the test. Only healthy fish free from observable diseases and abnormalities were used in the study.
- Feeding: Commercially available trout feed pellets with a diameter of 4 mm (e.g. Milkivit® Type F-2P B40, Skretting) were fed to the fish prior to the experimental phase. The same feed was used for the biomagnification experiments (spiked with test item).

HOLDING WATER:
- De-chlorinated local tap water in accordance with the OECD guideline (tap water sourced from the Schmallenberg district water production plants, mostly fed by small springs and percolation)
- The purification process included filtration with activated charcoal, passage through a lime-stone column, and aeration to the point of oxygen saturation
- To avoid copper contamination, plastic water pipes were used in the test facilities
- Chemistry data were recorded and reported monthly: temperature, pH, conductivity [µS cm-1], dissolved oxygen content [% ASV or mg O2 L-1], total residual chlorine [mg L-1], content of nitrate [mg L-1], nitrite [mg L-1], ammonium [mg L-1], phosphate [mg L-1], magnesium [mmol L-1], total hardness [mmol L-1], calcium hardness [mmol L-1], alkalinity [mmol L-1], NPOC content [mg L-1], and metal content (cadmium, chromium, copper, iron, manganese, nickel, lead, and zinc) [µg L-1].

Route of exposure:

feed

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Total depuration duration:

28 d

Hardness:

5.5 - 6.5 d° (mmol/L)

Test temperature:

9.9-16.7 °C

pH:

7.53 (mean value)

Dissolved oxygen:

8.65 (O2 mg/L) (mean value)

TOC:

0.641-1.554 mg/ L

Salinity:

n.a.

Conductivity:

255.0-272.0 µS/cm

Details on test conditions:

EXPERIMENTAL DESIGN:
Oncorhynchus mykiss with a total body length of 8 ± 4 cm at the start of the study, were used in the experiment. A test population of 57 fish was exposed under flow-through conditions for a period of 28 days to a concentration of 1000 mg kg-1 bis(2-ethylhexyl tetrabromophthalate (TBPH) via spiked feed, followed by a depuration phase of 28 days. An equally sized control population with fish of the same age and rise was monitored and sampled congruently. The test concentration and the duration of the uptake phase was chosen based on results of a pre- test, assessing two test concentrations (100 mg kg-1 and 1000 mg kg-1) over a course of 28 days uptake and another 28 days depuration. As no adverse effects were measurable under both conditions in the pre-test, the main study was conducted with the higher concentration. Test and control populations were both fed at a fixed ratio of 2 % of body weight per day. A maximum fish-to-water loading rate of 0.1 to 1.0 g fish (wet weight) per liter of water per day was not exceeded throughout the test by maintaining a continuous flow of at least 15.6 L h-1 water with a metering pump system. The experiment was conducted in 100 L glass aquaria, filled with 75 L holding and dilution water (control and treatment) at a scheduled temperature of 15 ± 2°C. The oxygen saturation in water was kept above 60% throughout the test by applying aeration via a glass capillary connected to an air pump. The vessels were left open and were subjected to a light/dark cycle of 16/8 hours. The oxygen concentration (WTW InoLab OXI 7310), temperature, and pH (WTW InoLab pH 7310) of the holding and dilution water was monitored daily in each vessel. TOC including organic carbon from particles and dissolved organic carbon, were measured as NPOC (Non-Purgeable Organic Carbon) before the test

OBSERVATIONS AND BIOLOGICAL MEASUREMENTS:
Fish of both groups, control and treatment, were sampled twice during the uptake phase (days 14 and 28) and five times during depuration (days 3.5, 7, 14, 21 and 28). Samplings were conducted before the daily feeding routine of the remaining test population to obtain samples from fasted fish. At each sampling event, six specimen of both populations were sacrificed at respective time points, recording whole body parameters (weight, length) and collecting the matrix samples (whole animal) after killing the fish humanly for analytical analyses. In addition, three specimen of each population were subjected to the same sampling procedures for monitoring lipid contents at the end of uptake and depuration phase for BMF lipid correction.
For all samplings fish were anesthetized in MS 222, rinsed, blotted dry and shock-frozen in liquid nitrogen after recording animal parameters. Sample material was stored at < -18°C until further processing. Each fish was analyzed individually for the test item to generate six replicates per sampling date. Control fish sampled at test start, end of uptake and end of depuration were first analysed with only one replicate of the sampled specimen to monitor contaminations. Further replicates as well as samples from other time points were considered, when test item was detected in controls.
Lipid fractions were determined by analyzing 3 individuals of each test population at the end of the uptake and depuration phase to calculate a lipid corrected BMF. In addition, the stock population was analysed as well.

QUALITY CONTROL:
The quality control samples were generated by adding 100 µL of each spike solution and 100 µL of the MTBPH spike solution into separate sample vials prepared with 800 µL acetonitrile to reach concentrations of 5 µg/L, 50 g/L, respectively 200 µg/L. The quality control samples were run alongside the sample batch to insure the applicability of the calibration.

Nominal and measured concentrations:

652 mg/kg nominal test concentration equals 38.9 mg/kg measured concentration (757 mg/kg (sample weight: 0.514 g))

Reference substance (positive control):

yes

Remarks:

Internal reference item MTBPH, Lot No: MBEHTBP0813, Purity: >98 %, Expiry date: June 10, 2020 (Wellington Laboratories Inc., see CoA in in A.6)

Lipid content:

>= 4.71 - <= 7.77 %

Time point:

other: End of uptake after 28 days

Remarks on result:

other: Fish lipid content

Lipid content:

>= 7.21 - <= 8.22 %

Time point:

other: End of depuration after 56 days

Remarks on result:

other: Fish lipid content

Lipid content:

>= 17.59 - <= 17.93 %

Time point:

other: end of uptake after 28 days

Remarks on result:

other: Feed lipid content

Lipid content:

>= 17.73 - <= 17.86 %

Time point:

other: End of depuration after 56 days

Remarks on result:

other: Feed lipid content

Key result

Conc. / dose:

652 other: mg/kg ww fish feed

Temp.:

ca. 15 °C

pH:

7.5

Type:

BMF

Value:

0.038 dimensionless

Basis:

total lipid content

Time of plateau:

56 d

Calculation basis:

other: growth- and lipid corrected

Conc. / dose:

652 other: mg/kg ww fish feed

Temp.:

ca. 15 °C

pH:

7.5

Type:

BMF

Value:

0.005 dimensionless

Basis:

total lipid content

Time of plateau:

56 d

Calculation basis:

kinetic

Conc. / dose:

652 other: mg/kg ww fish feed

Temp.:

ca. 15 °C

pH:

7.5

Type:

BMF

Value:

0.014 dimensionless

Basis:

total lipid content

Time of plateau:

56 d

Calculation basis:

kinetic, corrected for growth

Elimination:

yes

Depuration time (DT):

56 d

Rate constant:

growth rate constant (d-1)

Value:

0.029

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

0.015

Details on results:

Data evaluation:
Numerical values in this report are frequently rounded to a smaller degree of precision (number of digits) than used in the actual calculation. Minor differences in results obtained from calculations with such rounded values in comparison to those obtained with higher precision values are possible. They are, however, well within the limits of the experimental accuracy and thus of no practical concern.

Reported statistics:

The kinetic bioconcentration parameters were calculated accordingly to the suggested mathematical operations given in the OECD TG 305 for BMF determination using Microsoft Excel 2016® and SigmaPlot®.
 

Fish health and behavior:

All fish were in a vivid and healthy condition before and after the start of the experiment. On day 5 one fish was found in the outer chamber (for temperature regulation), originating from the exposure vessel. This fish was humanly euthanized and excluded from the sample material. Similarly, on day 39 one fish of each vessel (control and exposure, determined after counting remaining fish) was found in the outer chamber, but already dead. All remaining fish showed no abnormal behavior during the study. According to OECD TG 305 the mortality or other adverse effects in control and treated fish were less than 10 % at the end of the test.

Concentration of test item in diets:

The concentration of test item in control and test feed was assessed via LC-MS/MS analysis after spiking and extraction. The achieved concentration of test item on test feed was 652 µg/kg, while the control diet did not contain any trace of test item (values all < LOQ) after solvent-only spiking. The homogeneity of the feed with TBPH was tested in 5 replicates before test start. The low relative standard deviation of 2.3% (required: 15 %) indicated a successful evenly distribution of test item on feed. The test item on feed was also stable over time, as indicated by the contents of TBPH on feed at the end of the depuration phase with a recovery of 119.9% of the initial concentration (required ± 20%).

Concentration of test item in fish:

The concentration of test item in fish was assessed by chemical analysis via LC-MS/MS. The LOQ for TBPH in fish was validated with 10 µg kg-1. Fish were sampled at respective time points during the test as specified above. The stock population was free of a pre-contamination with the test item, as a representative control was sampled before test start, which had a concentration <LOQ. Sample concentrations of TBPH in fish matrix displayed a broad data range with values of 2820 µg/kg to 5876 µg kg-1 on day 14, and 4837 µg kg-1 to 11020 µg kg-1 on day 28 during the uptake phase. Both time points displayed a high relative standard deviation, with 31.8 % and 29.4 % on day 14 and day 28, respectively. This may be due to the uneven individual gut contents of spiked feed when fish were sampled. The reach of a steady-state concentration of test item in the uptake phase could not be drawn or neglected from the data. During depuration, data variation decreased (e.g. lower standard deviations 9.31 % up to 24.6%, which further supports the hypothesis that variable gut contents biased the data distribution during uptake.

During uptake, TBPH was detected in control fish >LOQ. Means in controls were 37.3 µg kg-1 and 32.1 µg kg-1 on day 14 and 28, which corresponds to 0.936 % and 0.374 % of the mean concentrations in the respective exposure groups.

Control fish monitored from the depuration phase displayed no contamination (days 31.5 and 56). The small amounts that were found may be due to carryover effects due to handling procedures as the aquaria were placed next to each other during experimentation. Nevertheless, this finding does not affect the validity of the study, since the stock control sample approved that fish were not pre-exposed or contaminated.

The results from the depuration phase allowed to determine the depuration rate constant k_2 with a linear fit (R2 = 0.8407) of the ln-transformed data. The respective depuration constant k_2 of 0.044 d-1 was determined from the slope. Based on k_2, the The results from the depuration phase allowed to determine the depuration rate constant k_2 with a linear fit (R2 = 0.8407) of the ln-transformed data. The respective depuration constant k_2 of 0.044 d-1 was determined from the slope. Based on k_2, the substance specific half-life is was determined with t_(1/2)= 15.61 days (uncorrected for growth).

Growth correction:

Fish were measured and weighted at the beginning of the experiment and at respective sampling time points to monitor growth and associated growth-dilution effects during experimentation. This physiological parameter is also an important measure to detect potential adverse effects that may occur with differing diets. Growth rate constants were determined separately for the uptake and depuration phases, for the treatment and control group, using the ln-transformed weights of the fish. A subsequent Parallel Line Analysis resulted in no statistical differences between the uptake and depuration phase among control animals (P = 0.6141), as well as for the TBPH-treated group (P = 0.4268). When analysing the slopes from depuration phase of control and treated group, no statistical difference was found (P = 0.9769). Considering all weight data (uptake and depuration), no statistically significant difference was detectable between control and the TBPH-treated group (P = 0.7905). Hence, it was deduced that no adverse, or toxic effects were caused by the diet with test item and that weight data of uptake and depuration phase of the treatment group can be pooled to derive the overall fish growth rate constant kg. The value determined for k_g of 0.0294 d-1 (R2 = 0.760), based on the pooled Ln-transformed weight data of the treated fish, was then used for growth correction of the depuration rate constants as recommended by OECD TG 305. The resulting growth-corrected depuration rate constant k_2g for TBPH was determined to be 0.0150 d-1. The growth-corrected half-life (t_(1/2g)) for TBPH was 46.24 days.

Lipid correction:

The lipid content of fish from the stock at the start of the experiment was about 4.5%. Over the experimental phase of the study, a mean lipid content of 6.8%was determined in control animals and 6.7% in TBPH-exposed specimen. The increase compared to the stock population was due to differing of feeding rates, with which fish were fed in culture (approx. 1% body weight per day), or during experimentation (2% feed of body weight per day). Fish feed contained 17.5% lipids in control diet and 17.8% in TBPH-spiked diet, respectively. The used feed fulfilled the recommendation of the OECD TG 305 with a content of 15-20% lipid in diets.

Validity criteria fulfilled:

yes

Conclusions:

After 28 days for uptake, and another 28 days for depuration a kinetic BMFk of 0.004 was determined subsequently, which was corrected for growth dilution effects resulting in a BMFkg of 0.014. Finally, a lipid-correction of the BMF was conducted, in order to standardize the BMF for comparative analyses of the results. The resulting BMFkgL of 0.038 indicates that bis(2-ethylhexyl tetrabromophthalate (TBPH) is not bioaccumulative to fish of the species Oncorhynchus mykiss when applied orally and uptake results via the digestive tract. The critical threshold for classification of substances to be bioaccumulative are BMF values >1 as suggested in the guidance document of the European commission for delivering environmental standards.

Executive summary:

To determine the biomagnification factor of bis(2-ethylhexyl) tetrabromophthalate (TBPH) in the fish species Oncorhynchus mykiss the test item was administered to a test population of fish via food in accordance to the conditions of the OECD TG 305 for BMF studies. A test concentration of 652 µg/kg was achieved by applying solvent-mediated spiking on regular feed pellets. In addition, untreated feed was used for a control population of fish to monitor potential mortalities or other adverse effects over time. The timeline of the test comprised 28 days for uptake, and another 28 days for depuration. During testing, no mortalities connected to the experiment were detected. Repeated samplings were done for both fish populations, removing 6 individuals at each sampling event for chemical analyses. During uptake phase, fish were sampled twice at days 14 and 28. Mean tissue concentrations were about approx. 4 mg/kg at day 14 and approx. 8.6 mg/kg at day 28 with high relative standard deviations of 31.8 % and 29.4 %, respectively. From five samplings during depuration, a depuration rate constant k₂ of 0.044 was calculated from the elimination dynamics of TBPH in fish matrix. Based on a (calculated) concentration of test item in fish of C_0,d approx. 2.2 mg/kg at the beginning of the depuration phase, an assimilation efficiency α of 0.011 was determined. A kinetic BMF_k of 0.004 was determined subsequently, which was corrected for growth dilution effects resulting in a BMF_kg =0.014. Finally, a lipid-correction of the BMF was conducted, in order to standardize the BMF for comparative analyses of the results. The resulting BMF_kgL of 0.038 indicates that TBPH is not bioaccumulative to fish of the species Oncorhynchus mykiss when applied orally and uptake results via the digestive tract. The critical threshold for classification of substances to be bioaccumulative are BMF values >1 as suggested in the guidance document of the European commission for delivering environmental standards.

Description of key information

After 28 days for uptake, and another 28 days for depuration a kinetic BMF_k of 0.004 was determined subsequently, which was corrected for growth dilution effects resulting in a BMF_kg of 0.014. Finally, a lipid-correction in order to standardize the BMF for comparative analyses of the results resulted in a BMF_kgL value of 0.038.

This result is supported by another valid BMF study (Nacci 2018), also performed according to OECD 305. The reported BMF values were in the range >=0.017 to <=0.024.

Key value for chemical safety assessment

BMF in fish (dimensionless):

0.038

Additional information

The resulting BMF_kgL of 0.038 indicates that TBPH is not bioaccumulative to fish of the species Oncorhynchus mykiss when applied orally and uptake results via the digestive tract. The critical threshold for classification of substances to be bioaccumulative are BMF values >1 as suggested in the guidance document of the European commission for delivering environmental standards.