Tetrabutylammonium bromide

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

Justification for type of information:

Experment performed using standard OECD test guidelines

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 2nd October, 2012

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1730 (Fish Bioconcentration Test)

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Vehicle:

no

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Source: Commercial fish farm, Manimangalam
- Sex: Male
- Age at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean, range and SD): Total length of 2.5 to 3.2 cm
- Weight at study initiation (mean and range, SD): 0.73505 g and 0.7237 g (for conc. 0.1 and 1.0 mg/l)
- Weight at termination (mean and range, SD): Depuration phase = 0.78255 g and 0.797 g (for conc. 0.1 and 1.0 mg/l)
- Lipid content at test initiation (mean and range, SD): 0.02956 g and 0.02893 g at 0.1 mg/l and 1.0 mg/l, respectively.
- Feeding during test : yes, test fishes were fed daily throughout the study period.
- Food type: Test fishes were fed with an appropriate diet of known lipid and total protein content in an amount sufficient to keep them in a healthy condition and to maintain body weight.
- Amount: Test fishes were fed throughout on a sufficient diet.
- Frequency: Daily
- Species identification: Fish species identification was done externally at Fisheries College and Research Institute (FCRI), Ponneri, Tiruvallur District, Tamil Nadu

ACCLIMATION
- Acclimation period: yes, the stock population of test fishes were acclimatized for 2 weeks.
- Acclimation conditions (same as test or not): yes, test conditions were same as the test.
- Type and amount of food: Test fishes were fed throughout on a sufficient diet of known lipid and total protein content in an amount sufficient to keep them in a healthy condition and to maintain body weight.
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): During the acclimatization period, there were no mortality observed for 14 days and hence, the batch was accepted.

Route of exposure:

aqueous

Test type:

semi-static

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Total depuration duration:

14 d

Hardness:

139 to 140 mg/l as CaCO3

Test temperature:

23.4 to 25°C

pH:

7.39 to 7.97

Dissolved oxygen:

84.3 to 102.7%

TOC:

< 5 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass tanks
- Material, size, headspace, fill volume: Glass aquaria (glass tank) of 30 lit capacity was used as a test vessel.
- Renewal rate of test solution (frequency/flow rate): yes, test medium was renewed thrice in a week and durng the renewal. fish from each group was transferred to a new medium in which test conc. are exposed in a test chamber.
- No. of organisms per vessel: 36 fishes/vessel for control and 72 fishes/vessel for each test group (0.1 and 1.0 mg/l)
- No. of vessels per concentration (replicates): 4 replicates
- No. of vessels per control / vehicle control (replicates): 4 replicates
- Biomass loading rate: 0.1 to 1.0 g/l
- Other: The leftout food and faeces were siphoned daily from the test chambers shortly after feeding (30 mins to 1 hr). Test chambers were kept clean as possible throughout the test to keep the conc. of organic matter as low as possible to avoid the presence of organic carbon that may limit the bioavailability of the test chemical.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The blended water was used in the study. It was prepared by mixing the reverse osmosis water and well water in the ratio of 3:1. This water was used during the acclomatization, preliminary experiment, uptake and depuration phase. The pH, hardness, total solids, total organic carbon (TOC), anions, cations, heavy metals and the presence of pesticides were analyzed.

OTHER TEST CONDITIONS
- Photoperiod:
Uptake phase- 12: 12 light:dark conditions controlled by an automatic timer.
Depuration phase- 6:6 light:dark conditions
- Light intensity:
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall daily feeding rate used in the study:
- For OECD 305 part III (dietary exposure fish bioaccumulation), number of feeds per day (number of feeds daily ration split between):
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall lipid content of spiked food before test start taking into account the contribution from the corn or fish oil vehicle, if used:
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall lipid content of spiked food after end of exposure taking into account the contribution from the corn or fish oil vehicle, if used:

RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: Test chemical conc. used for the study were 0.1 and 1.0 mg/l.
- Results used to determine the conditions for the definitive study: yes, preliminary study was carried out. A limit test was conducted at 100 mg/l alongwith control for 96 hrs. 7 fishes were exposed to 100 mg/l for a period of 96 hrs. An equal control was also maintained. No mortality or toxicity signs were observed during the study period upto 96 hrs.
The highest conc. (1 mg/l) of the test chemical was selected base on 1% of its acute LC50 (100 mg/l). The second conc. (0.1 mg/l) was selected from the one above by factor of 10. Thus, exposure conc. were 0.1 and 1.0 mg/l, respectibely.
- Other justification for choice of test concentrations:

Nominal and measured concentrations:

Test chemical conc. used for the study were 0.1 and 1.0 mg/l

Lipid content:

29.56 mg/kg bw w.w.

Time point:

start of exposure

Remarks on result:

other: at 0.1 mg/l during uptake phase

Lipid content:

28.93 mg/kg bw w.w.

Time point:

start of exposure

Remarks on result:

other: at 1.0 mg/l during uptake phase

Lipid content:

35.9 mg/kg bw w.w.

Time point:

start of exposure

Remarks on result:

other: at 0.1 mg/l during depuration phase

Lipid content:

35.41 mg/kg bw w.w.

Time point:

end of exposure

Remarks on result:

other: at 1.0 mg/l during depuration phase

Key result

Conc. / dose:

0.1 mg/L

Temp.:

> 23.4 - < 25 °C

Type:

BCF

Value:

0.839 dimensionless

Basis:

whole body w.w.

Time of plateau:

28 d

Remarks on result:

other: pH = 7.39 to 7.97

Key result

Conc. / dose:

1 mg/L

Temp.:

> 23.4 - < 25 °C

Type:

BCF

Value:

0.462 dimensionless

Basis:

whole body w.w.

Time of plateau:

28 d

Remarks on result:

other: pH = 7.39 to 7.97

Details on results:

- Mortality of test organisms:
During the acclimatization period, there was no mortaliy observed and fish was found to be healthy.
In preliminary test: there was no mortalirty or toxicity signs in test fishes.

Details of depuration and rate constant was provided in the attached document of IUCLID.

No mortality and toxicity signs (abnormal behaviour) was observed in the studies exposure conc. 0.1 and 1.0 mg/l and in control during the observation period of both uptake and depuration phases.

Linearity

Linearity curve was constructed peak area counts versus concentration of standards (ng/mL) showed the correlation co-efficient is 1.000, The method was found to be linear as

indicated by the correlation co-efficient value. The calibration data are presented in Table 4 and the calibration curve is presented in Figure 1. The representative chromatograms are presented from Figures 23 and 24.

System Precision

The repeatability of the system was confirmed by the observed relative standard deviation (RSD) concentration of 1.0 ng/mL. The results showed the RSD (%) was within the

acceptable limit. The respective data are presented in Table 5. The representative chromatogram is presented in Figure 25.

Assay Accuracy

The analytical method shows the acceptable mean percent recovery of Tetrabutylammonium bromide in medium were 91% + 3.62 at 0.01 mg/L fortification level; 98 % + 6.63 at 0.1 mg/L fortification level. The control samples showed no significant interference.

The analytical method shows the acceptable mean percent recovery of Tetrabutylammonium bromide in fish were 84 % + 5.74 at 0.01 mg/kg fortification level; 85 % + 4.02 at 0.1 mg/kg fortification level. The control samples showed no significant interference. The respective data are presented from Tables 6 - 7. The representative chromatograms are presented from Figures 26 - 31.

Quantification

Uptake phase

During the uptake phase, the fish were exposed at the concentration of 0.1 mg/L and 1.0 mg/L in water for 28 days at 20-25 °C. The results showed the average. Tetrabutylammonium bromide content in fish was Day 0 - 0.012 mg/kg, Day 7 - 0.0514 mg/kg, Day 14 - 0.0718 mg/kg, Day 21 - 0.0781 mg/kg and Day 28 - 0.0831 mg/kg in the 0.1 mg/L exposed concentration. The active content in medium was analyzed for 0.1 mg/L concentration and the results were Day 0 — 0.1092 mg/L, Day 7 ~ 0.1048 mg/L, Day 14 -— 0.1081 mg/L, Day 21 — 0.1095 and Day 28 - 0.0990 mg/L. Analysis of 1.0 mg/L exposed fish samples showed the average Tetrabutylammonium bromide content in fish Day 0 — 0.0621 mg/kg, Day 7 — 0.2419 mg/kg, Day 14 - 0.4050 mg/kg, Day 21 — 0.4338 mg/kg and Day 28 — 0.4641 mg/kg. The active content in medium was analyzed for 0.1 mg/L concentration and the results were Day 0 — 0.9323 mg/L, Day 7 — 0.9188 mg/L, Day 14 — 0.9386 mg/L, Day 21 - 0.8784 and Day 28 — 1.0036 mg/L. The uptake phase rate constant (k:) calculated was 0.0610 and 0.0658 for 0.1 mg/L and 1.0 mg/L concentrations respectively. The results are presented in Tables 12 - 21 and Tables 30 - 32. The uptake phase curve is presented in Figures 2 - 4. The representative chromatograms are presented in Figures 35-45 and Figures 55 - 65.

Depuration phase

During the depuration phase, control and test group (0.1 mg/L and 1.0 mg/L) fish were released into pure medium (without test item) separately for 14 days at 20-25 °C. The

results showed the average Tetrabutylammonium bromide content in fish was Day 1 — 0.0582 mg/kg, Day 3 — 0.0221 mg/kg, Day 7 and Day 14 were below determination level

(<LOQ) in the 0.1 mg/L exposed fish concentration. The active content in medium was analyzed for 0.1 mg/L concentration and the residues of tetrabutylammonium bromide were

below determination in all the occasions (Day 0, Day 1, Day 7 and Day14). Analysis of 1.0 mg/L exposed fish samples showed the average Tetrabutylammonium bromide content

in fish Day 1 ~ 0.3197 mg/kg, Day 3 — 0.1211 mg/kg, Day 7 — 0.0454 mg/kg and Day 14 - 0.0174 mg/kg. The active content in medium was analyzed for 1.0 mg/L concentration and the residues of Tetrabutylammonium bromide were below determination in all the occasions (Day 0, Day 1, Day 7 and Dayl4). The depuration phase rate constant (k2) calculated was -0.4842 and -0.2113 for 0.1 mg/L and 1.0 mg/L concentrations respectively.

The results are presented in Tables 22 - 29 and Tables 33 - 34. The depuration phase curve was presented in Figures 5-6. The representative chromatograms are presented in Figures 46 - 54 and Figures 66 - 74,

Growth Rate Constant

The individual fish weight data are converted to In(weight). Curve was plotted In(weight) versus time (day), separately for control and test groups. The same process is carried out for uptake and depuration phases separately. The results are presented from Tables 36 - 42 and Figures 7 - 12.

Lipid Content

The lipid content of the fish was analyzed for before the uptake phase, end of the uptake and end of the depuration phase. The before uptake phase analysis samples showed the

lipid content as 0.02956g and 0.02893g for exposed fish at 0.1 mg/L and 1.0 mg/L concentrations respectively. The end of uptake phase analysis samples showed the lipid

content as 0.03405g and 0.03054g for exposed fish at 0.1 mg/L and 1.0 mg/L concentrations respectively. At the depuration phase analysis samples showed the lipid

content as 0.03590g and 0.03541g for exposed fish at 0.1 mg/L and 1.0 mg/L concentrations respectively. The mean 5% lipid normalized content was 0.0205 mg/Kg and 0.1073 for 0.1 mg/L and 1.0 mg/L in the uptake phase at day 0. In end of the uptake phase lipid content was 0.1219 mg/kg and 0.7597 mg/kg respectively. In depuration phase 0.0246 mg/kg for 1.0 mg/L. Lipid content was within 25 % deviation in control and test group. The results are presented in Table 35.

Bio-Concentration Factor (BCF)

Uptake phase

The steady state concentration of the test substance in fish (Ci) was 0.0831 mg/kg and 0.4641 mg/kg for 0.1 mg/L and 1.0 mg/L concentrations respectively. The concentration of

the test substance in water medium (Cy) was 0.0990 mg/kg and 1.0036 mg/kg for 0.1 mg/L and 1.0 mg/L concentrations respectively at steady state. The bio-concentration

factor (BCF) was 0.8394 for 0.1 mg/L concentration and 0.4624 for 1.0 mg/L concentrations respectively. The results are presented in Table 43

Kinetic Bio-Concentration Factor (BCFk)

The overall uptake rate constant (k:) was 0.0610 for 0.1 mg/L concentration and 0.0658 for 1.0 mg/L concentration. The overall depuration rate constant (kz) was -0.4842 for 0.1 mg/L concentration and -0.2113 for 1.0 mg/L concentrations. The kinetic bio-concentration factor (BCF) of Tetrabutylammnium bromide was -0.1261 for 0.1 mg/L concentration and 0.3114 for 1.0 mg/L concentration. The results are presented in Table 44.

Growth-corrected kinetic Bio-Concentration factor (BCFkg)

The depuration growth rate constant (kg) was 0.0080 for 0.1 mg/L and -0.0039 for 1.0 mg/L concentrations. The growth-corrected depuration rate constant (kz) was -0.4922 for

0.1 mg/L and -0.2074 for 1.0 mg/L concentrations. The overall uptake rate constant (k1) was 0.0610 for 0.1 mg/L concentration and 0.0658 for 1.0 mg/L concentration. The

growth-corrected kinetic bio-concentration factor (BCFkg) of Tetrabutylammnium bromide was -0.1240 for 0.1 mg/L concentration and -0.3173 for 1.0 mg/L concentration. The

results are presented in Table 45.

Lipid normalized kinetic Bio-Concentration Factor (BCFkl)

The average 5% lipid normalized content (Ly) analysis of day 0 was 0.02956 g at 0.1 mg/L and 0,02983 g at 1.0 mg/L in uptake phase. Analysis of lipid content in end the uptake was 0.03405 g and 0.03054g, The lipid normalized kinetic bio-concentration factor of (BCFkl) was -0.2133 and -05382 for 0.1 mg/L and 1.0 mg/L respectively at start of uptake phase and end of the uptake was -0.1852 and -0.5097 for 0.1 mg/L and 1.0 mg/L. The results are presented in Table 46.

Validity criteria fulfilled:

yes

Conclusions:

The Bio-concentration factor (BCF) was determined as 0,8394 and 0.4624 for 0.1 mg/L and 1.0 mg/L concentrations respectively in the uptake phase. Further, based on the overall rate constants of the uptake and depuration phases, the kinetic Bio-concentration factor (BCFk) was determined to be -0.1261 and -0.3114 for the concentrations 0.1 mg/L and 1.0 mg/L respectively. From the growth rate analysis, the growth corrected kinetic bio-concentration factor (BCFkg) was determined to be -0.1240 and -0.3173 for the concentrations 0.1 mg/L and 1.0 mg/L respectively. Based on the lipid content analysis, the lipid normalized kinetic bio-concentration factor (BCFkl) was determined to be -0.1852 and -0.5097 for the concentrations 0.1 mg/L and 1.0 mg/L respectively in the uptake phase.

Executive summary:

Bioaccumulation study in fish was conducted for determining the bioconcentration factor of test chemical. The study was performed in accordance with the OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test and EPA OPPTS 850.1730 (Fish Bioconcentration Test), respectively. Danio rerio (Zebra fish) (male) of total length 2.5 to 3.2 cm obtained from commercial fish farm, Manimangalam was used as a test fishes. Fish used in the tests were free from observable diseases and abnormalities. Test fishes were fed daily throughout the study period. Fishes were fed with an appropriate diet of known lipid and total protein content in an amount sufficient to keep them in a healthy condition and to maintain body weight. Fish species identification was done externally at Fisheries College and Research Institute (FCRI), Ponneri, Tiruvallur District, Tamil Nadu. Acclimatization of the stock population of test fishes were done for 2 weeks. Test conditions were same as the test. Feeding to test fishes were done in the same manner as that done during the study. Preliminary study was carried out. A limit test was conducted at 100 mg/l alongwith control for 96 hrs. 7 fishes were exposed to 100 mg/l for a period of 96 hrs. An equal control was also maintained. No mortality or toxicity signs were observed during the study period upto 96 hrs. The highest conc. (1 mg/l) of the test chemical was selected base on 1% of its acute LC50 (100 mg/l). The second conc. (0.1 mg/l) was selected from the one above by factor of 10. Thus, exposure conc. were 0.1 and 1.0 mg/l, respectibely. The blended water was used in the study. It was prepared by mixing the reverse osmosis water and well water in the ratio of 3:1. This water was used during the acclomatization, preliminary experiment, uptake and depuration phase. The pH, hardness, total solids, total organic carbon (TOC), anions, cations, heavy metals and the presence of pesticides were analyzed.Glass aquaria (glass tank) of 30 lit capacity was used as a test vessel. Test chemical conc. used for the study were 0.1 and 1.0 mg/l. Total 72 fishes/vessel for each test group were exposed with the test chemical. Study was performed under semi-static conditions and test medium was renewed thrice in a week and durng the renewal. fish from each group was transferred to a new medium in which test conc. are exposed in a test chamber. Biomass loading rate during the study was 0.1 to 1.0 g/l, respectively. One control was run in addition to the test series. A control group of fish was held under experimental conditions except for the absence of the test chemical. The leftout food and faeces were siphoned daily from the test chambers shortly after feeding (30 mins to 1 hr). Test chambers were kept clean as possible throughout the test to keep the conc. of organic matter as low as possible to avoid the presence of organic carbon that may limit the bioavailability of the test chemical. The blended water was used in the study. It was prepared by mixing the reverse osmosis water and well water in the ratio of 3:1. This water was used during the acclomatization, preliminary experiment, uptake and depuration phase. The pH, hardness, total solids, total organic carbon (TOC), anions, cations, heavy metals and the presence of pesticides were analyzed. Test conditions involve a pH of 7.39 to 7.97, hardness of 139 to 140 mg/l as CaCO3, temperature of 23.4 to 25°C, dissolved oxygen of 84.3 to 102.7%, TOC of < 5 mg/l, respectively. Uptake phase was carried out for 28 d and depuration phase for 14 d, respectively. Photoperiod during the uptake phase was 12: 12 light:dark conditions controlled by an automatic timer and depuration phase was 6:6 light:dark conditions, respectively. All experiments were performed in triplicates.The leftout food and faeces were siphoned daily from the test chambers shortly after feeding (30 mins to 1 hr). Test chambers were kept clean as possible throughout the test to keep the conc. of organic matter as low as possible to avoid the presence of organic carbon that may limit the bioavailability of the test chemical. After removal of lipid content from the sample, the aqueous layer was transferred into 50 mL centrifuge tube and the volume was adjusted to 10 mL using acetonitrile. The sample was homogenized for 2 minutes using tissumizer. The sample solution was centrifuged at 3000 rpm and decanted, A 2 mL of sample solution was filtered through 0.2 um PTFE and analysed for active content. The final solution was filtered and analyzed by LC-MS/MS conditions. HPLC system consists of Agilent 1290 Infinity UHPLC, Agilent Zorbax Eclipse XDB (2.1 mm X 75 mm, 3.5 μm), column temperature of 40°C, injection volume of 10 μl, flow rate of 1.0 ml/min, auto dampler temp. of 10°C. Gradient conditions involve the use of 0.1% acetic acid in 90/10 (MilliQ water/ Methanol) and 0.1%  acetic acid in acetonitrile, respectively. Total run time was 9.0 mins with a retention time of 1.5 mins. MS conditions involves the detector of Agilent 6490 LC-MS/MS System, coupled with Mass Hunter instrument control and data processing software, AJS-ESI interface, Positive polarity, Gas temperature of 260°C, Gas flow of 16 L/min, Nebulizer gas flow of 50 Psi, Sheath gas temperature of 400°C, Sheath gas flow of 11 L/min, Capillary voltage of 3500V, CAV of 4 V and Dwell time of 200 sec., respectively. During the uptake and depuration phase, the fish samples were extracted immediately and analyzed for active content. The uptake phase rate constants (k1), depuration phase rate constant (k2) was calculated by plotting the Ln (comc. of fish, mg/kg) Vs occasions (days). During the acclimatization period, there was no mortaliy observed and fish was found to be healthy. In preliminary test: there was no mortalirty or toxicity signs in test fishes. During the study, the water temperature variation was less than + 2°C which ranged from 23.4 to 25.0°C, the concentration of dissolved oxygen had not been fallen below 60% saturation and the range was 84.3 to 102.7%, the concentration of the test item (0.1 & 1.0 mg/L) in the chambers was maintained within + 20% of the mean of the measured values during the uptake phase, the concentration of the test item (0.1 & 1.0 mg/L) was below its limit of solubility in water, taking into account the effect that the test water may have on effective solubility, no mortality and other adverse effect was found in both control and treated fish during the uptake and depuration phases. No significant differences in average growth between the test and the control groups of sampled fish (Hence there was no indication of toxic effect of the test chemical, thereby fulfilling the validity criteria and hence, study was considered to be valid.The Bio-concentration factor (BCF) was determined as 0,8394 and 0.4624 for 0.1 mg/L and 1.0 mg/L concentrations respectively in the uptake phase. Further, based on the overall rate constants of the uptake and depuration phases, the kinetic Bio-concentration factor (BCFk) was determined to be -0.1261 and -0.3114 for the concentrations 0.1 mg/L and 1.0 mg/L respectively. From the growth rate analysis, the growth corrected kinetic bio-concentration factor (BCFkg) was determined to be -0.1240 and -0.3173 for the concentrations 0.1 mg/L and 1.0 mg/L respectively. Based on the lipid content analysis, the lipid normalized kinetic bio-concentration factor (BCFkl) was determined to be -0.1852 and -0.5097 for the concentrations 0.1 mg/L and 1.0 mg/L respectively in the uptake phase. Since, the BCF value did not exceeded the threshold limit of 2000, thus, test chemical was considered to be non-bioaccumulative,

Description of key information

Bioaccumulation study in fish was conducted for determining the bioconcentration factor of test chemical. The study was performed in accordance with the OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test and EPA OPPTS 850.1730 (Fish Bioconcentration Test), respectively. Danio rerio (Zebra fish) (male) of total length 2.5 to 3.2 cm obtained from commercial fish farm, Manimangalam was used as a test fishes. Fish used in the tests were free from observable diseases and abnormalities. Test fishes were fed daily throughout the study period. Fishes were fed with an appropriate diet of known lipid and total protein content in an amount sufficient to keep them in a healthy condition and to maintain body weight. Fish species identification was done externally at Fisheries College and Research Institute (FCRI), Ponneri, Tiruvallur District, Tamil Nadu. Acclimatization of the stock population of test fishes were done for 2 weeks. Test conditions were same as the test. Feeding to test fishes were done in the same manner as that done during the study. Preliminary study was carried out. A limit test was conducted at 100 mg/l alongwith control for 96 hrs. 7 fishes were exposed to 100 mg/l for a period of 96 hrs. An equal control was also maintained. No mortality or toxicity signs were observed during the study period upto 96 hrs. The highest conc. (1 mg/l) of the test chemical was selected base on 1% of its acute LC50 (100 mg/l). The second conc. (0.1 mg/l) was selected from the one above by factor of 10. Thus, exposure conc. were 0.1 and 1.0 mg/l, respectibely. The blended water was used in the study. It was prepared by mixing the reverse osmosis water and well water in the ratio of 3:1. This water was used during the acclomatization, preliminary experiment, uptake and depuration phase. The pH, hardness, total solids, total organic carbon (TOC), anions, cations, heavy metals and the presence of pesticides were analyzed.Glass aquaria (glass tank) of 30 lit capacity was used as a test vessel. Test chemical conc. used for the study were 0.1 and 1.0 mg/l. Total 72 fishes/vessel for each test group were exposed with the test chemical. Study was performed under semi-static conditions and test medium was renewed thrice in a week and durng the renewal. fish from each group was transferred to a new medium in which test conc. are exposed in a test chamber. Biomass loading rate during the study was 0.1 to 1.0 g/l, respectively. One control was run in addition to the test series. A control group of fish was held under experimental conditions except for the absence of the test chemical. The leftout food and faeces were siphoned daily from the test chambers shortly after feeding (30 mins to 1 hr). Test chambers were kept clean as possible throughout the test to keep the conc. of organic matter as low as possible to avoid the presence of organic carbon that may limit the bioavailability of the test chemical. The blended water was used in the study. It was prepared by mixing the reverse osmosis water and well water in the ratio of 3:1. This water was used during the acclomatization, preliminary experiment, uptake and depuration phase. The pH, hardness, total solids, total organic carbon (TOC), anions, cations, heavy metals and the presence of pesticides were analyzed. Test conditions involve a pH of 7.39 to 7.97, hardness of 139 to 140 mg/l as CaCO3, temperature of 23.4 to 25°C, dissolved oxygen of 84.3 to 102.7%, TOC of < 5 mg/l, respectively. Uptake phase was carried out for 28 d and depuration phase for 14 d, respectively. Photoperiod during the uptake phase was 12: 12 light:dark conditions controlled by an automatic timer and depuration phase was 6:6 light:dark conditions, respectively. All experiments were performed in triplicates.The leftout food and faeces were siphoned daily from the test chambers shortly after feeding (30 mins to 1 hr). Test chambers were kept clean as possible throughout the test to keep the conc. of organic matter as low as possible to avoid the presence of organic carbon that may limit the bioavailability of the test chemical. After removal of lipid content from the sample, the aqueous layer was transferred into 50 mL centrifuge tube and the volume was adjusted to 10 mL using acetonitrile. The sample was homogenized for 2 minutes using tissumizer. The sample solution was centrifuged at 3000 rpm and decanted, A 2 mL of sample solution was filtered through 0.2 um PTFE and analysed for active content. The final solution was filtered and analyzed by LC-MS/MS conditions. HPLC system consists of Agilent 1290 Infinity UHPLC, Agilent Zorbax Eclipse XDB (2.1 mm X 75 mm, 3.5 μm), column temperature of 40°C, injection volume of 10 μl, flow rate of 1.0 ml/min, auto dampler temp. of 10°C. Gradient conditions involve the use of 0.1% acetic acid in 90/10 (MilliQ water/ Methanol) and 0.1%  acetic acid in acetonitrile, respectively. Total run time was 9.0 mins with a retention time of 1.5 mins. MS conditions involves the detector of Agilent 6490 LC-MS/MS System, coupled with Mass Hunter instrument control and data processing software, AJS-ESI interface, Positive polarity, Gas temperature of 260°C, Gas flow of 16 L/min, Nebulizer gas flow of 50 Psi, Sheath gas temperature of 400°C, Sheath gas flow of 11 L/min, Capillary voltage of 3500V, CAV of 4 V and Dwell time of 200 sec., respectively. During the uptake and depuration phase, the fish samples were extracted immediately and analyzed for active content. The uptake phase rate constants (k1), depuration phase rate constant (k2) was calculated by plotting the Ln (comc. of fish, mg/kg) Vs occasions (days). During the acclimatization period, there was no mortaliy observed and fish was found to be healthy. In preliminary test: there was no mortalirty or toxicity signs in test fishes. During the study, the water temperature variation was less than + 2°C which ranged from 23.4 to 25.0°C, the concentration of dissolved oxygen had not been fallen below 60% saturation and the range was 84.3 to 102.7%, the concentration of the test item (0.1 & 1.0 mg/L) in the chambers was maintained within + 20% of the mean of the measured values during the uptake phase, the concentration of the test item (0.1 & 1.0 mg/L) was below its limit of solubility in water, taking into account the effect that the test water may have on effective solubility, no mortality and other adverse effect was found in both control and treated fish during the uptake and depuration phases. No significant differences in average growth between the test and the control groups of sampled fish (Hence there was no indication of toxic effect of the test chemical, thereby fulfilling the validity criteria and hence, study was considered to be valid.The Bio-concentration factor (BCF) was determined as 0,8394 and 0.4624 for 0.1 mg/L and 1.0 mg/L concentrations respectively in the uptake phase. Further, based on the overall rate constants of the uptake and depuration phases, the kinetic Bio-concentration factor (BCFk) was determined to be -0.1261 and -0.3114 for the concentrations 0.1 mg/L and 1.0 mg/L respectively. From the growth rate analysis, the growth corrected kinetic bio-concentration factor (BCFkg) was determined to be -0.1240 and -0.3173 for the concentrations 0.1 mg/L and 1.0 mg/L respectively. Based on the lipid content analysis, the lipid normalized kinetic bio-concentration factor (BCFkl) was determined to be -0.1852 and -0.5097 for the concentrations 0.1 mg/L and 1.0 mg/L respectively in the uptake phase. Since, the BCF value did not exceeded the threshold limit of 2000, thus, test chemical was considered to be non-bioaccumulative,

Key value for chemical safety assessment

BCF (aquatic species):

0.839 dimensionless

Additional information

Bioaccumulation study in fish was conducted for determining the bioconcentration factor of test chemical. The study was performed in accordance with the OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test and EPA OPPTS 850.1730 (Fish Bioconcentration Test), respectively. Danio rerio (Zebra fish) (male) of total length 2.5 to 3.2 cm obtained from commercial fish farm, Manimangalam was used as a test fishes. Fish used in the tests were free from observable diseases and abnormalities. Test fishes were fed daily throughout the study period. Fishes were fed with an appropriate diet of known lipid and total protein content in an amount sufficient to keep them in a healthy condition and to maintain body weight. Fish species identification was done externally at Fisheries College and Research Institute (FCRI), Ponneri, Tiruvallur District, Tamil Nadu. Acclimatization of the stock population of test fishes were done for 2 weeks. Test conditions were same as the test. Feeding to test fishes were done in the same manner as that done during the study. Preliminary study was carried out. A limit test was conducted at 100 mg/l alongwith control for 96 hrs. 7 fishes were exposed to 100 mg/l for a period of 96 hrs. An equal control was also maintained. No mortality or toxicity signs were observed during the study period upto 96 hrs. The highest conc. (1 mg/l) of the test chemical was selected base on 1% of its acute LC50 (100 mg/l). The second conc. (0.1 mg/l) was selected from the one above by factor of 10. Thus, exposure conc. were 0.1 and 1.0 mg/l, respectibely. The blended water was used in the study. It was prepared by mixing the reverse osmosis water and well water in the ratio of 3:1. This water was used during the acclomatization, preliminary experiment, uptake and depuration phase. The pH, hardness, total solids, total organic carbon (TOC), anions, cations, heavy metals and the presence of pesticides were analyzed.Glass aquaria (glass tank) of 30 lit capacity was used as a test vessel. Test chemical conc. used for the study were 0.1 and 1.0 mg/l. Total 72 fishes/vessel for each test group were exposed with the test chemical. Study was performed under semi-static conditions and test medium was renewed thrice in a week and durng the renewal. fish from each group was transferred to a new medium in which test conc. are exposed in a test chamber. Biomass loading rate during the study was 0.1 to 1.0 g/l, respectively. One control was run in addition to the test series. A control group of fish was held under experimental conditions except for the absence of the test chemical. The leftout food and faeces were siphoned daily from the test chambers shortly after feeding (30 mins to 1 hr). Test chambers were kept clean as possible throughout the test to keep the conc. of organic matter as low as possible to avoid the presence of organic carbon that may limit the bioavailability of the test chemical. The blended water was used in the study. It was prepared by mixing the reverse osmosis water and well water in the ratio of 3:1. This water was used during the acclomatization, preliminary experiment, uptake and depuration phase. The pH, hardness, total solids, total organic carbon (TOC), anions, cations, heavy metals and the presence of pesticides were analyzed. Test conditions involve a pH of 7.39 to 7.97, hardness of 139 to 140 mg/l as CaCO3, temperature of 23.4 to 25°C, dissolved oxygen of 84.3 to 102.7%, TOC of < 5 mg/l, respectively. Uptake phase was carried out for 28 d and depuration phase for 14 d, respectively. Photoperiod during the uptake phase was 12: 12 light:dark conditions controlled by an automatic timer and depuration phase was 6:6 light:dark conditions, respectively. All experiments were performed in triplicates.The leftout food and faeces were siphoned daily from the test chambers shortly after feeding (30 mins to 1 hr). Test chambers were kept clean as possible throughout the test to keep the conc. of organic matter as low as possible to avoid the presence of organic carbon that may limit the bioavailability of the test chemical. After removal of lipid content from the sample, the aqueous layer was transferred into 50 mL centrifuge tube and the volume was adjusted to 10 mL using acetonitrile. The sample was homogenized for 2 minutes using tissumizer. The sample solution was centrifuged at 3000 rpm and decanted, A 2 mL of sample solution was filtered through 0.2 um PTFE and analysed for active content. The final solution was filtered and analyzed by LC-MS/MS conditions. HPLC system consists of Agilent 1290 Infinity UHPLC, Agilent Zorbax Eclipse XDB (2.1 mm X 75 mm, 3.5 μm), column temperature of 40°C, injection volume of 10 μl, flow rate of 1.0 ml/min, auto dampler temp. of 10°C. Gradient conditions involve the use of 0.1% acetic acid in 90/10 (MilliQ water/ Methanol) and 0.1%  acetic acid in acetonitrile, respectively. Total run time was 9.0 mins with a retention time of 1.5 mins. MS conditions involves the detector of Agilent 6490 LC-MS/MS System, coupled with Mass Hunter instrument control and data processing software, AJS-ESI interface, Positive polarity, Gas temperature of 260°C, Gas flow of 16 L/min, Nebulizer gas flow of 50 Psi, Sheath gas temperature of 400°C, Sheath gas flow of 11 L/min, Capillary voltage of 3500V, CAV of 4 V and Dwell time of 200 sec., respectively. During the uptake and depuration phase, the fish samples were extracted immediately and analyzed for active content. The uptake phase rate constants (k1), depuration phase rate constant (k2) was calculated by plotting the Ln (comc. of fish, mg/kg) Vs occasions (days). During the acclimatization period, there was no mortaliy observed and fish was found to be healthy. In preliminary test: there was no mortalirty or toxicity signs in test fishes. During the study, the water temperature variation was less than + 2°C which ranged from 23.4 to 25.0°C, the concentration of dissolved oxygen had not been fallen below 60% saturation and the range was 84.3 to 102.7%, the concentration of the test item (0.1 & 1.0 mg/L) in the chambers was maintained within + 20% of the mean of the measured values during the uptake phase, the concentration of the test item (0.1 & 1.0 mg/L) was below its limit of solubility in water, taking into account the effect that the test water may have on effective solubility, no mortality and other adverse effect was found in both control and treated fish during the uptake and depuration phases. No significant differences in average growth between the test and the control groups of sampled fish (Hence there was no indication of toxic effect of the test chemical, thereby fulfilling the validity criteria and hence, study was considered to be valid.The Bio-concentration factor (BCF) was determined as 0,8394 and 0.4624 for 0.1 mg/L and 1.0 mg/L concentrations respectively in the uptake phase. Further, based on the overall rate constants of the uptake and depuration phases, the kinetic Bio-concentration factor (BCFk) was determined to be -0.1261 and -0.3114 for the concentrations 0.1 mg/L and 1.0 mg/L respectively. From the growth rate analysis, the growth corrected kinetic bio-concentration factor (BCFkg) was determined to be -0.1240 and -0.3173 for the concentrations 0.1 mg/L and 1.0 mg/L respectively. Based on the lipid content analysis, the lipid normalized kinetic bio-concentration factor (BCFkl) was determined to be -0.1852 and -0.5097 for the concentrations 0.1 mg/L and 1.0 mg/L respectively in the uptake phase. Since, the BCF value did not exceeded the threshold limit of 2000, thus, test chemical was considered to be non-bioaccumulative,