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Environmental fate & pathways

Bioaccumulation: aquatic / sediment

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Reference
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 21 February 2017 and 17 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 305 B (Bioaccumulation: Semi-static Fish Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Test substance: GL500
Chemical name: 1,4-Benzenedicarboxylic acid, mixed Bu and 2-ethylhexyl diester (CAS name) Reaction product of terephthalic acid, 2-ethylhexanol and 1-butanol (IUPAC name)
Molecular formula: A: C16H22O4 B: C19H30O4 C: C24H38O4
Molecular weight: A: 278 B:322 C: 390
CAS No.: 1571954-81-8 (A: 1962-75-0 B:1429441-82-6 C: 6422-86-2)
Purity: 99.8%
Lot number: GLFG160607
Water solubility: 2.19 mg/L (20 °C)
Appearance: Colorless liquid
Expiry date: 2017-06-01
Storage conditions: Room temperature (25 oC), no contact with air and light
Radiolabelling:
not specified
Details on sampling:
Sampling and Analysis of Fish and Water
(1) Fish and water sampling schedule
Water from the test chambers were sampled for the determination of test substance concentration before addition of the fish and during the first 0h, 24h, 48h, 72h at the beginning of the test.
Water from the test chambers were sampled for the determination of test substance concentration during uptake phases. At the beginning of the test 0d, and exposure phase 22, 23, 24, 25, 26, 27, 28d, the water was sampled at the same time as the fish and before feeding.
(2) Sampling and sample treatment
Water samples for analysis were obtained by siphoning through inert tubing from a central point in the test chamber. Seven fish were removed from the test chambers at each sampling time. The sampled fish were rinsed quickly with water, blotted “dry”, killed instantly, using the most appropriate and humane method, and then weighed.
Treatment of extraction and concentration for the fish samples: seven fish and some anhydrous sodium sulfate were put in the mortar and ground. The samples were extracted by adding 10.0 mL dichloromethane and extracted. The process was repeated twice and the extract solutions combined. The extract solutions were dried by rotary evaporation and then re-dissolved by 10.0 mL acetonitrile. The final solution was injected to UPLC-PDA after being filtered with 0.22 μm millipore membrane.
Treatment of extraction and concentration for the water sample: Water sample (20.0 mL) was extracted by adding 10.0 mL dichloromethane and extracted. The process was repeated twice and the extract solutions combined. The extract solutions were dried by rotary evaporation and then re-dissolved by 20.0 mL acetonitrile. The final solution was injected to UPLC-PDA after being filtered with 0.22 μm membrane.
The quantitative calculation was made according to the peak areas of control blank and test solutions.
(3) Analytical method
A. Preparation of standard stock solution
The standard stock solution I of the test substance (1000 mg/L) was prepared by dissolving 0.0500 g test substance into 50.0 mL acetonitrile.
The standard stock solution II of the test substance (100 mg/L) for the calibration curve was prepared by diluting 10 mL of storage solution I with acetonitrile to a total volume of 100 mL.
The standard stock solution III of the test substance (10.0 mg/L) for the calibration curve was prepared by diluting 10.0 mL of storage solution II with acetonitrile to a total volume of 100 mL.
B. Working solution
The working solution of 0.20, 0.50, 1.00, 2.00, 5.00, 10.0, 20.0 mg/L were prepared with acetonitrile.
Details of the working solutions are showed as follows:
Concentration
(mg/L) Concentration of the Stock Solution Added (mg/L) Volume of the Stock Solution Added
(mL) Final Volume after Dilution
(mL)
0.20 10.0 0.200 10.0
0.50 10.0 0.500 10.0
1.00 10.0 1.00 10.0
2.00 10.0 2.00 10.0
5.00 10.0 5.00 10.0
10.0 10.0 10.0 10.0
20.0 100 2.00 10.0
C. UPLC-PDA condition:
Equipment: ACQUITY™ Ultra Performance LC (Waters USA)
Chromatographic column: ACQUITY UPLC® BEH C18 1.7 μm 2.1×10 mm Column (Waters USA)
Detector: ACQUITY UPLC PDA Detector
Wavelength: 242 nm
Column temperature: 30C
Mobile phase: acetonitrile: water=90:10
Flow rate: 0.300 mL/min
Inject volume: 10 μL
Under the above conditions, the retention times of test substance were about 1.8, 3.5, 8.2 min.
Vehicle:
no
Details on preparation of test solutions, spiked fish food or sediment:
Preparation of the Test Solutions
According to the acute toxicity information provided by sponsor (96 h-LC50 > 100 mg/L WAFs), and the solubility of test substance (2.19 mg/L, 20ºC), the test solutions were prepared with saturated solution and its 10% dilutions.
The aqueous saturated solution of test substance was prepared via adding test substance (~ 0.5 g) in 20 L test water at 35ºC for 24h, height of vortex was less than 8% of liquid surface static height, when saturation is achieved the mixture is cooled at 23ºC, subsequently, the mixture is filtered by glass fiber filter membrane. The initial filtrates (about 100 mL) were discarded to minimize the adsorption effect. The filtration was treated as a stock solution (saturated solution), and the solution was clear and colorless. The test solutions were stock solution and another solution diluted by the stock solution. 2 parallels were set for both test solutions. A control blank was set at the same time.
Details for the test solutions preparation were described as following
Concentration Stock solution (L) Total volume (L)
Control blank 0 20
10% stock solution 2.0 20
100% stock solution 20 20
Test organisms (species):
other: Rare minnow (Gobiocypris rarus)
Details on test organisms:
Test Fish
(1) Selection of species
Important criteria in the selection of species are that they are readily available, can be obtained in convenient sizes and can be satisfactorily maintained in the laboratory.
The test adopted Rare minnow (Gobiocypris rarus) (Batch No.: F20170115G) were bred by our own lab. All fishes in the test were from one single population.
(2) Holding of fish
The stock population of fish was acclimated for 15 days in water at the test temperature and feed throughout on a sufficient diet (1%~2% weight of fish) and of the same type to be used during the test. Following a 48-hour settling-in period, no fish died during the holding time of 7 d. The fish used in tests were free from observable diseases and abnormalities. Fish did not receive any treatment for disease in the two weeks preceding the test, and during the test.
The ingredients of the fish food are given as follows: Crude Protein > 36.0%, Crude Fat > 2.0%, Crude Fibre < 3.0%, Crude Ash < 12.0%, Moisture < 10.0%.
The ingredients of the fish food and concentrations of pollutants are determined by Jiangsu Provincial Center for Disease Control and Prevention, 2 times per year at least.

Water for Holding/Diluting
Good quality tap water which had been dechlorinated for at least 24 hours was used as holding water. The test species can survive, grow and reproduce in the holding water and no abnormal appearance or behaviour appeared.
The water for diluting has the same source. The water quality parameters are determined by Jiangsu Provincial Center for Disease Control and Prevention, 2 times per year at least
Route of exposure:
aqueous
Test type:
semi-static
Water / sediment media type:
other: Good quality tap water which had been dechlorinated for at least 24 hours
Total exposure / uptake duration:
28 d
Hardness:
140 to 149 mg/L CaCO3
Test temperature:
22.9 to 23.4 ºC
pH:
7.69 to 8.07
Dissolved oxygen:
71.8 to 99.9 %
Details on test conditions:
Conditions of Exposure
(1) Renewal of the test solution
During the test, aeration was not used. The time interval of changing exposure solutions was 72 hours under the requisition of quality assurance and quality control. When changing the test solution, transfer of the fish to the fresh medium of the test substance was performed quickly. The fresh test solution was prepared 24 h in advance to ensure the balance of test substance and water.
(2) Numbers of test fish and fish loading
All the test fish used had the same source and age. The mean length of fishes was 3.0±0.5 cm and the mean weight was 0.2±0.1 g. The test fish were randomly assigned to the treatment levels and controls with 70 fish each 20 L tank. The loading rate was 0.7 g of fish (wet weight) per litre of water.
(3) Feeding
During the acclimation and test periods, an appropriate diet of known lipid and total protein content was fed to the fish in an amount sufficient to keep them in a healthy condition and to maintain body weight. The fish were fed daily throughout the acclimation and test periods at a level of approximately 1% to 2% of body weight per day. Uneaten food and faeces were siphoned daily from the test chambers shortly after feeding (30 minutes to 1 hour). The chambers were kept as clean as possible throughout the test so that the concentration of organic matter was kept as low as possible.
(4) Light and temperature
During the test, the following conditions were maintained:
-Light: 16 hours photoperiod daily;
-Temperature: (23±1) ºC.
Nominal and measured concentrations:
According to the acute toxicity information provided by sponsor (96 h-LC50 > 100 mg/L WAFs), and the solubility of test substance (2.19 mg/L, 20ºC), the test solutions were prepared with saturated solution and its 10% dilutions.
Details on estimation of bioconcentration:
(1)BCF
A sample of water and fish was taken each day in parallel after 0, 22, 23, 24, 25, 26, 27, 28 days. Therefore, 8 concentration values of water samples and fish samples were obtained and then 8 BCF values were calculated.
The uptake curve of the test substance was obtained by plotting its concentration in/on fish in the uptake phase against time on arithmetic scales after 0d. The steady state BCFss is calculated from the following equation:

BCFss = Cf(steady state)/Cw (steady state)

Cf: mean concentration of test substance in fish sample;
Cw: mean concentration of test substance in water sample.

Otherwise, the concentration factor is expressed as the range of BCF value.
However, since the test substance concentration in fish didn’t reach equilibrium, the BCF28 was used to represent biological concentration factor of fish.

(2) Relative Standard Deviation (RSD)
Use the “STEDV” function of MS® Office Excel to calculate the SD, RSD is the ratio of the SD against to average of the concentration:

RSD(%) = (SD/C) * 100

Where:
RSD: Relative Standard Deviation (%);
SD: Standard Deviation (%);
C: Average of concentration.
Key result
Conc. / dose:
>= 51.6 - <= 56.3
Type:
BCF
Value:
>= 116 - <= 139 other: mg/kg
Basis:
not specified
Time of plateau:
28 d
Calculation basis:
steady state
Remarks on result:
other: 100% stock solution
Key result
Conc. / dose:
>= 47.6 - <= 56.6
Type:
BCF
Value:
>= 10.8 - <= 13 other: mg/kg
Basis:
not specified
Time of plateau:
28 d
Calculation basis:
steady state
Remarks on result:
other: 10% saturated solution
Details on kinetic parameters:
During the test (22~28d), for 100% stock solution set, the measured concentration of samples in water was 2.21~2.49 mg/L (mean 2.38 mg/L, i.e. saturated concentration); and the measured concentration of samples in fish was 116~139 mg/kg (mean 128 mg/kg). Therefore, for the set, BCF of the test substance was 51.6~56.3.
For 10% stock solution set, the measured concentration of samples in water was 0.213~0.237 mg/L (mean 0.227 mg/L, i.e. 10% saturated concentration); and the measured concentration of samples in fish was 10.8~13.0 mg/kg (mean 12.0 mg/kg). Therefore, for the set, BCF of the test substance was 47.6~56.6.
Details on results:
During the test (22~28d), for 100% stock solution set, the measured concentration of samples in water was 2.21~2.49 mg/L (mean 2.38 mg/L, i.e. saturated concentration); and the measured concentration of samples in fish was 116~139 mg/kg (mean 128 mg/kg). Therefore, for the set, BCF of the test substance was 51.6~56.3.
For 10% stock solution set, the measured concentration of samples in water was 0.213~0.237 mg/L (mean 0.227 mg/L, i.e. 10% saturated concentration); and the measured concentration of samples in fish was 10.8~13.0 mg/kg (mean 12.0 mg/kg). Therefore, for the set, BCF of the test substance was 47.6~56.6.
Conclusions:
The results show that BCF reach the steady state at the 22nd~28th day. Therefore, according to the concentrations for water and fish, BCFss (95% confidence interval) for fish was 52.9 ±5 .71 (47.6~56.6, 10% saturated solution) and 53.5 ± 2.99 (51.6~ 56.3, 100% saturated solution).
Executive summary:

According to “The guidelines for the testing of chemicals” (HJ/T 153-2004) and “The Guidelines for the Testing of Chemicals, Degradation and Accumulation” and with reference to Procedure 305B of the “Guidelines for Testing of Chemicals”of the OECD: Bioconcentration: Semi-static Fish Test(OECD TG 305B), the bioconcentration factor (BCFs) of the test substance in fish (Gobiocypris rarus) was tested.

The results showed that the recovery in the water sample was 106% and 106%, and RSD was 2.72% and 2.18%, when the added concentration of the test substance is 0.20 mg/L and 20.0 mg/L in water samples, respectively. The recovery in the fish sample was 111%and 101%, RSD was 2.08% and 0.57%, while the added concentration of the test substance is12.5mg/kg and 100 mg/kg in fish samples, respectively. The limit of detection (LOD) is 0.06 mg/L, and the limit of quantity(LOQ) is 0.20 mg/L for apparatus based on S/N≥ 3 andS/N≥ 10. The LOQs for water sample and fish sample are 0.20 mg/L and 2.00 mg/kg respectively.

The results show that theBCFssvalues of test substance were 52.9 and 53.5, when the concentration exposed to is 10% saturated solution (measured concentration 0.227 mg/L) and 100% saturated solution (measured concentration 2.38 mg/L).

Description of key information

The results show that BCF reach the steady state at the 22nd~28th day. Therefore, according to the concentrations for water and fish, BCFss (95% confidence interval) for fish was 52.9 ±5 .71 (47.6~56.6, 10% saturated solution) and 53.5 ± 2.99 (51.6~ 56.3, 100% saturated solution).

Key value for chemical safety assessment

BCF (aquatic species):
53.5 dimensionless

Additional information