Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

30 November 2018 to 08 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

OECD study in accordance with current legislation.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guidelines for Testing of Chemicals, 305-III: Dietary Exposure Bioaccumulation Fish Test. Paris: OECD. Adopted: 2 October 2012.

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes

Specific details on test material used for the study:

No further details specified in the study report.

Radiolabelling:

no

Details on sampling:

Water: The water samples were collected at the 7d, 11d, 14d, 21d, 24d, 28d, and 30d of the uptake phase to analysis the concentration of test item. Two samples were taken from the test group and one from the control.
Fish: The fish samples were collected from the control and the test group at the same days as described above to analysis the concentration of the test item. At least 2 fish in control and 10 to 15 fish (in replicates) in test group were taken for measurement before feeding.

Vehicle:

no

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

Although the test item is liquid, due to the low concentration (200mg/kg) of exposure and the small amount (10-159) of diets prepared, if without the dispersion of acetone, the weighing amount of 2 mg- 3 mg is too small to satisfy the accuracy requirement of the balance and also leads to uneven dispersion. So, acetone was used to mix the test item with the diet. 32.0 mg test item was accurately weighed and dissolved in acetone to make stock solution with concentration of 1280 mg/L.
The preparation of diet: added a certain volume of the stock solution to a certain amounts of fish feed to make the diet with concentration of 200 mg/kg, mixed it well, took it in the ventilation cabinet to allow the excess solvent to evaporate naturally. The next day, three samples were randomly selected, and the concentration of the samples was measured. The control group was treated with the same amount of acetone (without the test item).

Test organisms (species):

other: Rare minnow (Gobiocypris rarus).

Details on test organisms:

Test organisms: Rare minnow (Gobiocypris rarus). The fish were of approximately the same size and age (i.e. the length of the largest fish is not more than two-thirds of that of the smallest fish). The length of fish was in the range of 3.0-4.0 cm. The body weight was in the range of 0.4-0.69. The fish were healthy and free from any apparent malformation or behavioral change.
Justification for selection: Rare minnow ( Gobiocypris rarus) is easy to obtain and breed, and is quite sensitive to various chemicals. It is widely used in fish toxicity testing and has rich data available for reference purpose. It is used as a representative model organism in China. It can be used to carry the test of 305-ID: Dietary Exposure Bioaccumulation Fish Test.
Source: Rare minnow (Code No.: Rare minnow C20180118) were reproduced and maintained in the lab of CTI-SET after introduced in 2014 from the Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China.
Acclimation: the fish were acclimated in the water with the same quality of test water 2 weeks before the test. During the acclimation, the temperature was controlled between 21 to 25°C, within the range of ± 2°C, the light was maintained for 12 hours daily, and the oxygen concentration was maintained above 80% of air saturation value. The fish were fed every day. The total mortality of the fish was 0%, which was less than the required 5% maximum mortality during the 7 days acclimation period at the beginning of the test, the fish could be used.

Route of exposure:

feed

Justification for method:

dietary exposure method used because stable, measurable water concentrations cannot be maintained

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

30 d

Total depuration duration:

0 d

Hardness:

190 mg/L (in CaCO3)

Test temperature:

22.2~23.8 °C

pH:

pH value of the water was in the range of 7.64 - 7.96.

Dissolved oxygen:

99.0%~100.6%ASV

TOC:

1.02 mg/L

Salinity:

Not applicable - freshwater study

Conductivity:

Not specified

Details on test conditions:

Preliminary Test
As the test item is a UVCB substance, four principal component ions were measured by LC-MS/MS to characterize the concentration of the test.
In the pre-test of acute toxicity, 0.4, 0.8 and 1.6 mg/L were tested for aqueous exposure and concentration analysis. The result showed that after 96 hours of exposure, the mortality offish was 10% at 0.4 mg/Land 100% at 0.8 mg/Land 1.6 mg/L respectively. However, in the concentration of 0.4 mg/L, the test item could not maintained in the range of 80%~120% of the initial, and reduced to about half after exposure of 24 hours, which was consistent with the result of the fish acute toxicity test (Study Number: 511883) provided by the sponsor.
In order to determine the stability of the test item at the low exposure level of bioaccumulation test (should be no more than 2% of the LC50), the two concentration levels of 5 μg/L and 50 μg/L were monitored for potential changes in concentration for 24 hours. By comparing the responses and changes of the components at 0, 6 and 24 h, the concentration could not be maintained for 6 hours.
The above results showed that neither semi-static nor flow-through system can be ensured to keep the concentration within the range required by the test by the way of aqueous exposure, therefore, only dietary exposure can be used.
The diets with concentrations of 1000 and 200 mg/kg were prepared through dietary exposure. The feeding amount was 1.5% of the body weight of the fish.- In the 1000 mg/kg group, all fish demonstrated slow eating at the beginning of exposure, and refused to eat after 48 hours and showed erratic swimming. After 72 hours, the fish were lethargic, and after 96 hours hyperventilation and imbalance was observed. 40% mortality was found after 120 hours. After 7 days of exposure, the survived fish were sampled to analyze the concentration of test item. The result showed that no test item was detected in the concentration of 1000 mg/kg possibly due to the food refusal of the exposed fish.
The exposure group of 1000 mg/kg was terminated then. The fish fed 200 mg/kg concentration were normal during the exposure period.
During the test, the accuracy, homogeneity and stability of the fish feed prepared were investigated, and the time of steady state and the amount of bioaccumulation were calculated. The result showed that the accuracy deviation in the diet was 91.3%~118%, which was in the range of 80%-120% of nominal. The homogeneity result showed that the RSD was 2.2%~ 14.7%, less than the requirement of 15%.
Under the refrigerated condition, the test item in diet was stable for 19 days with a deviation of 10.5%, which was kept in the range of ±20% of the initial concentration.
In the 200 mg/kg group, the concentration of test item in the fish reached a relatively stable state after 12 days of exposure. The BMF value after 30 days was 8. 70E-5, which indicated that the bioaccumulative capacity of the test item in fish is very low, and it is not necessary to carry out the depuration.
According to the results of the range-finding test, main test was conducted with the concentration of 200 mg/kg in diet.

Exposure
The test exposure system was a flow-through exposure system. The test container was a 20 L fish tank with 50 fish. There were three tanks in the test group and one tank in the control. During the exposure, the flow rate was controlled about 1 00ml/min, with a renewal volume of 7 to 8 volumes of tank each day.

Observation and Measurement
Observation
During the test, the fish were observed daily to evaluate the number of mortalities and the number of individuals exhibiting signs of abnormal behavior. Water temperature was monitored every day, and pH and dissolved oxygen of water in all test containers were measured once a week; Hardness and total organic carbon of water were determined at the beginning of the test.

Measurement
Diet
At the uptake stage, before and after the test feed renewal and at the end of the uptake phase, 3 samples were randomly selected from the control and the test diets, and the concentration of the test item was measured.
The lipid contents of the control and test diets were measured before the start of the test and at the end of the uptake phase.

Fish
During the test, 20 fish at the beginning and 10 fish at the end were randomly selected from each group to measure body length and body weight.
At the beginning of uptake phase, 10 fish was randomly selected from the control and the test group to determine the lipid content. On the 24d, 28d and 30d (the end of the test), 10 fish was randomly selected to measure the lipid content respectively.
It can check if similar quantities of offered diet have been consumed by comparing the growth rate between the test and control groups. A t-test on growth rate constants was performed, to test whether growth differs between control and test groups.

Treatment of the Test System
At the end of the test, surviving fish were collected and placed in the aqueous solution of ethylene glycol phenyl ether (with concentration of 2%o-5%o) to be euthanized.
The dead fish were stored temporarily in the refrigerator and then delivered to the approved waste handling company for disposal.

Nominal and measured concentrations:

In the pre-test of acute toxicity, 0.4, 0.8 and 1.6 mg/L were tested for aqueous exposure and concentration analysis.
The test concentration of 200 mg/kg MLA-3202 was selected based on the results of the range finding test.

Reference substance (positive control):

no

Details on estimation of bioconcentration:

Treatment of Results
Calculation of BMFss
In the dietary exposure bioaccumulation fish test, if the concentration of the test item in the fish reaches a steady state during the uptake phase, the biomagnification factor of the steady state can be evaluated and calculated by the following formula:

BMFss = Cfss / Cfood

Where: BMFss-the steady-state biomagnification factor;
Cfss- Mean concentration of test item in fish at steady state (mg/kg);
Cfood - Mean concentration of test item in diet (mg/kg).

(2) Assumed Specific Growth Rate Calculation
The "assumed" specific growth rate is the single-tailed growth rate derived from the comparison of the initial average weight of the fish in the whole test vessel with the following formula:

r = ([InW2 – InW1] / [t2 – t1]) x 100

Where: r-Specific growth rate;
W1-The weight of a single fish at the start of the test;
W2-The weight of a single fish at the end of the test;
InW1-The mean logarithmic of body weight per fish at the start of the test;
InW2-The logarithm of the body weight per fish at the end of the test;
t1, t2-The interval between the start and the end of the test, day.

(3) Calculation of Lipid-Correction factor (Lc)

Lc = Lfish / Lfood

Where: Lc-Lipid-Correction Factor;
Lfish-the mean lipid fraction in fish (%);
Lfood -the mean lipid fraction in fish feed (%).

(4) Calculation of Lipid-Corrected steady-state biomagnification factor (BMFssL)

BMFssL = BMFss / Lc

Where: BMFssL-Lipid-corrected steady-state biomagnification factor;
BMFss-steady-state biomagnification factor;
Lc-Lipid-corrected factor.

Lipid content:

8.1 %

Time point:

start of exposure

Lipid content:

13.2 %

Time point:

end of exposure

Key result

Conc. / dose:

200 mg/g food

Temp.:

> 22.2 - < 23.8 °C

pH:

7.96

Type:

BMF

Value:

0.001 dimensionless

Basis:

other: Lipid-corrected steady state biomagnification factor.

Time of plateau:

30 d

Calculation basis:

steady state

Details on kinetic parameters:

Not determined

Metabolites:

Not determined

Details on results:

Water Quality
Total hardness and the total organic carbon (TOC) of the test water were measured as 190 mg/L (in CaCO3) and 1.02 mg/Lat the beginning of the test. The particulate matter was measured quarterly with the value of 0.6±0.2mg/L.
During the period of the exposure, water temperature was maintained in the range of 22.2-23.8°C. pH value was in the range of 7.64-7.96; dissolved oxygen was in the range of 99.0%-100.6% ASV.

Mortality and Abnormal Behavior of Fish
During the period of exposure, there was no mortality or abnormal behavior in the control group and test group.

Growth Rate
At the beginning of uptake phase, the average body weights of control and test group were 0.473 g ± 0.064 g and 0.496 g ± 0.085 g respectively; the average body lengths were 3.5 cm ± 0.1 cm and 3.6 cm ± 0.1 cm respectively. At the end of absorption phase, the average body weights of control and test group were 0.515 g ± 0.036 g and 0.536 g ± 0.014 g respectively; the average body lengths were 3.6 cm± 0.1 cm and 3.6 cm ± 0.2 cm respectively. It showed that the fish did not significantly grow during the test.
According to the results of assumed specific growth rate and t-test, there was no statistically different of specific growth rate between test group and control group (p=0.31 ). No adverse effects were observed on the growth of test fish with diet spiked with test item.

Analysis of Diets
During the uptake phase, five batches of diets were prepared, 3 samples of each batch were randomly selected from the control and test group for analysis. The measured concentration for prepared diet was 91.5%-119% of nominal. The RSD was 0.57%-2.6%, within the range of ±15%, so it showed high degree of homogeeity.
During the uptake phase, after stored, the test item in the expired diets was measured as 81.5%-109% of the new diets, the concentration of the test substance in fish food before and at the end of the uptake phase was within a range of ± 20%. The concentration during the uptake phase was calculated as 206.1 mg/kg by the geometric mean. No test item was detected in control diet (without test item).

Analysis of Fish
In the uptake phase, the measured average concentrations in the fish were in the range of 0.092 - 0.181 mg/kg; the test item was not detected in fish of control group.

Analysis of Test Water
In the uptake phase, the test item was not detected in test water of control group and test group.

Lipid Content
At the beginning of the uptake phase, the average lipid contents (Ltish) of control and test group were 8.6% and 8.1 % respectively. At 24d, 28d and 30d of absorption phase, the average lipid contents (Ltish) of test group were 10.4-10.7%.
At the beginning and the end of uptake phase, the average lipid contents (Ltood) of diet were both 13.2%.

The Steady-State Biomagnification Factor (BMFss/ BMFssL)
During the uptake phase, the concentration in fish was in the range of 0.092 mg/kg to 0.181 mg/kg with slight changes, indicating that steady-state conditions had been reached after 7 days. Considering that the lipid content in fish from 24 day to 30 day was determined to be 10.5%, which was relatively stable, according to the mean measured concentration in fish and the mean lipid content from 24d to 30d, the BMFss value was calculated as 6.987E-4, the BMFssL value was 8. 789E-4. Given the extremely low BMF, the depuration step was not undertaken.

Concentration Result of Acute Toxic Solutions (Range-Finding Test)

Nominal concentration (mg/L)	Replicate	Measured concentration (mg/L)								O/N
		0h (N)	24h (O)	24h (N)	48h (O)	48h (N)	72h (O)	72h (N)	96h (N)
0.4	1	0.47	0.23	0.47	0.20	0.39	0.18	0.31	0.20	49%
	2	0.46	0.23	0.49	0.19	0.42	0.18	0.31	0.20
	Mean	0.46	0.23	0.48	0.19	0.40	0.18	0.31	0.20
0.8	1	0.76	0.54	0.75	0.52	0.78	0.48	0.62	0.57	71%
	2	0.76	0.53	0.72	0.54	0.80	0.42	0.64	0.61
	Mean	0.76	0.53	0.74	0.53	0.79	0.45	0.63	0.59
1.6	1	1.48	1.11	1.40	1.14	1.65	1.15	1.29	1.15	77%
	2	1.44	1.11	1.41	1.06	1.67	1.16	1.36	1.17
	Mean	1.46	1.11	1.41	1.10	1.66	1.15	1.32	1.16

 

Results of Fish Feed Accuracy and Uniformity (Range-finding Test)

Diet batch	Date analyzed	Rep.	Diet (g)	Measured conc. (μg/L)	To volume (mL)	Con. In diet (μg/g)	Mean (μg/g)	Rec. %	RSD %
1000μg/g– 20180920-1	2018-09-26	1	0.534	102224.636	5.0	957.2	912.7	91.3	4.3
		2	0.515	92692.116	5.0	899.9
		3	0.475	83686.954	5.0	880.9
200μg/g-20180926-1	2018-09-26	1	0.502	10601.339	10.0	211.2	215.6	108	2.9
		2	0.511	11242.898	10.0	2220.0
200μg/g-20180930-1	2018-09-30	1	0.521	11423.163	10.0	219.3	212.4	106	3.6
		2	0.519	11102.018	10.0	213.9
		3	0.517	10549.383	10.0	204.0
200μg/g-20181008-1	2018-10-09	1	0.515	10863.271	10.0	210.9	216.3	108	2.2
		2	0.516	11299.200	10.0	219.0
		3	0.509	11152.285	10.0	219.1
200μg/g-20181010-1	2018-10-12	1	0.502	12440.599	10.0	247.8	217.2	109	13.0
		2	0.502	10637.422	10.0	211.9
		3	0.503	9654.856	10.0	191.9
200μg/g-20181011-1	2018-10-12	1	0.504	9855.911	10.0	195.6	235.4	118	14.7
		2	0.500	12774.834	10.0	255.5
		3	0.499	12728.641	10.0	255.1

Note: Rep. means Replicate; Con. means Concentration; Rec. means Recovery.

 

Stability of Test item in Diet (Range-finding Test)

Diet Batch	200μg/g-20180930-1
Date analyzed	Rep.	Diet (g)	Measured conc. (μg/L)	To volumes (mL)	Con. in diet (μg/g)	Mean (μg/g)	RSD%
2018-09-30	1	0.521	11423.163	10.0	219.3	212.4	/
	2	0.519	11102.018	10.0	213.9
	3	0.517	10549.383	10.0	204.0
2018-10-09 (9 d)	1	0.527	12267.161	10.0	232.8	223.3	5.12%
	2	0.508	10698.046	10.0	210.6
	3	0.517	11708.321	10.0	226.5
2018-10-18 (19 d)	1	0.495	9357.422	10.0	189.0	190.1	10.5%
	2	0.490	9381.795	10.0	191.5
	3	0.497	9440.002	10.0	189.9

Note: the stability deviation% = (average determination result in initial fish feed (μg/g)-average determination result in fish feed (μg/g) / average determination result in initial fish feed (μg/g) * 100%

 

Concentration in Fish under Dietary Exposure (Range-Finding Test)

Concentration	200mg/kg
Date analyzed	Day	Rep.	Fish (g)	Measured Con. (μg/L)	To volume (mL)	Con. in fish (mg/kg)	Mean (mg/kg)
2018-10-03	7d	1	1.968	105.307	10.0	0.535	0.541*
		2	1.968	107.538	10.0	0.546
2018-10-08	12d	1	2.401	4.804	10.0	0.020	0.020
		2	2.401	ND	10.0	NA
2018-10-12	16d	1	1.864	22.224	2.0	0.024	0.023
		2	1.864	20.757	2.0	0.022
2018-10-18	22d	1	1.759	18.408	2.0	0.021	0.021
		2	1.759	18.266	2.0	0.021
2018-10-23	27d	1	2.302	35.974	2.0	0.031	0.022
		2	2.242	13.258	2.0	0.012
2018-10-24	28d	1	2.170	16.142	2.0	0.015	0.011
		2	2.245	8.775	2.0	0.008
2018-10-26	30d	1	1.956	24.520	2.0	0.025	0.019
		2	2.004	13.265	2.0	0.013

Note: “ND” means not detected; “NA” means not applicable; “*” the fish was sampled and analyzed 3 hours after each feed, do the result showed that the dietary exposure was feasible

 

Temperature during the Exposure (Main Test)

Day after exposure	Temperature monitored in the flow-through system (°C)
	Min	Max
0d	22.5	23.2
1d	22.3	23.1
2d	22.4	23.4
3d	22.6	23.2
4d	22.7	23.5
5d	22.5	23.5
6d	22.8	23.7
7d	22.4	23.3
8d	22.3	23.2
9d	22.3	23.3
10d	22.2	23.3
11d	22.4	23.2
12d	22.7	23.5
13d	22.8	23.6
14d	22.9	23.5
15d	22.6	23.7
16d	22.8	23.5
17d	22.9	23.4
18d	22.4	23.5
19d	22.3	23.7
20d	22.4	23.6
21d	22.5	23.1
22d	22.5	23.3
23d	22.6	23.5
24d	22.6	23.7
25d	22.6	23.6
26d	22.5	23.7
27d	22.4	23.6
28d	22.5	23.6
29d	22.6	23.3
30d	22.7	23.8
Temperature range (°C)	22.2 – 23.8

 

pH value and Dissolved Oxygen (Main Test)

Day after exposure	pH		DO (%ASV)
	Control	200 mg/kg	Control	200 mg/kg
-1d	7.93	7.91	99.0	99.2
7d	7.89	7.92	100.1	100.6
14d	7.88	7.96	99.0	99.0
21d	7.82	7.91	99.6	99.8
24d	7.64	7.80	100.2	100.4
28d	7.77	7.85	99.5	99.0

 

Mortality and Abnormal Behavior (Main Test)

Day after exposure	Mortality and abnormal behavior
	Control	200 mg/kg
0d	0, A	0, A
1d	0, A	0, A
2d	0, A	0, A
3d	0, A	0, A
4d	0, A	0, A
5d	0, A	0, A
6d	0, A	0, A
7d	0, A	0, A
8d	0, A	0, A
9d	0, A	0, A
10d	0, A	0, A
11d	0, A	0, A
12d	0, A	0, A
13d	0, A	0, A
14d	0, A	0, A
15d	0, A	0, A
16d	0, A	0, A
17d	0, A	0, A
18d	0, A	0, A
19d	0, A	0, A
20d	0, A	0, A
21d	0, A	0, A
22d	0, A	0, A
23d	0, A	0, A
24d	0, A	0, A
25d	0, A	0, A
26d	0, A	0, A
27d	0, A	0, A
28d	0, A	0, A
29d	0, A	0, A
30d	0, A	0, A
Mortality	0%	0%

Note: “A” means no mortality and abnormal behavior

 

Growth Rate for Exposure Fish during the Test (Main Test)

Time (d)	Rep.	Control			200 mg/kg
		Weight (g)	Growth rate (%)	Length (cm)	Weight (g)	Growth rate (%)	Length (cm)
Up-0	1	0.511	/	3.5	0.504	/	3.6
	2	0.476	/	3.4	0.507	/	3.5
	3	0.411	/	3.4	0.511	/	3.7
	4	0.478	/	3.4	0.411	/	3.5
	5	0.478	/	3.6	0.482	/	3.5
	6	0.409	/	3.5	0.502	/	3.7
	7	0.415	/	3.5	0.455	/	3.5
	8	0.469	/	3.4	0.506	/	3.5
	9	0.487	/	3.4	0.507	/	3.6
	10	0.446	/	3.6	0.513	/	3.6
	11	0.477	/	3.4	0.500	/	3.6
	12	0.498	/	3.5	0.512	/	3.6
	13	0.482	/	3.4	0.498	/	3.5
	14	0.503	/	3.5	0.502	/	3.5
	15	0.500	/	3.5	0.503	/	3.5
	16	0.497	/	3.4	0.488	/	3.6
	17	0.510	/	3.6	0.511	/	3.6
	18	0.505	/	3.5	0.505	/	3.6
	19	0.462	/	3.4	0.502	/	3.5
	20	0.451	/	3.5	0.499	/	3.6
	Mean	0.473	/	3.5	0.499	/	3.6
	SD	0.032	/	0.073	0.024	/	0.067
Up-30	1	0.535	0.42	3.6	0.544	0.31	3.8
	2	0.521	0.33	3.7	0.532	0.24	3.6
	3	0.489	0.12	3.6	0.535	0.26	3.6
	4	0.503	0.21	3.6	0.522	0.17	3.7
	5	0.507	0.24	3.5	0.541	0.29	3.5
	6	0.534	0.41	3.6	0.543	0.31	3.6
	7	0.551	0.51	3.7	0.541	0.29	3.5
	8	0.524	0.35	3.7	0.532	0.24	3.7
	9	0.488	0.11	3.6	0.540	0.29	3.7
	10	0.496	0.16	3.5	0.531	0.23	3.6
	Mean	0.515	0.29	3.6	0.536	0.26	3.6
	SD	0.021	0.14	0.074	0.007	0.04	0.095
T-test		P=0.31

 

Homogeneity of the Diet (Main Test)

Nominal Concentration (mg/kg)	Date	Measured concentration (mg/kg)				Recovery (%)	RSD (%)
		1	2	3	Mean
200	181204	224.750	229.259	236.474	230.161	115	2.6
	181211	214.045	208.421	215.739	212.735	106	1.8
	181218	205.905	205.247	213.580	208.244	104	2.2
	181225	238.405	237.587	235.754	237.249	119	0.57
	190101	184.558	184.403	180.175	183.045	91.5	1.4

 

Measured Concentration in the Diet during the Test (Main Test)

Time (d)		Control				200 mg/kg				O / N (%)
		1	2	3	Mean	1	2	3	Mean
Up-0	N	ND	ND	ND	NA	224.8	229.3	236.5	230.2	85.9
Up-7	O	ND	ND	ND	NA	209.4	191.5	191.9	197.6
	N	ND	ND	ND	NA	214.0	208.4	215.7	212.7	96.9
Up-14	O	ND	ND	ND	NA	197.3	262.4*	215.0	206.1
	N	ND	ND	ND	NA	205.9	205.2	213.6	208.2	109
Up-21	O	ND	ND	ND	NA	220.5	227.0	236.3	227.9
	N	ND	ND	ND	NA	238.4	237.6	235.8	237.2	81.5
Up-28	O	ND	ND	ND	NA	188.5	203.8	187.9	193.4
	N	ND	ND	ND	NA	184.6	184.4	180.2	183.0	94.9
Up-30	O	ND	ND	ND	NA	165.4	179.1	176.8	173.7
Geometric mean		NA				206.1				NA

Note: “ND” means not detected; “NA” mean not applicable; “N” means new prepared diet; “O” means diet prepared after one week; “*” abnormal, eliminated.

 

Measured Concentration if Fish during the Test (Main Test)

Time (d)	Control	200 mg/kg
		1	2	Mean (mg/kg)
7	ND	0.107	0.011*	0.107
11	ND	0.105	0.080	0.092
14	ND	0.102	0.092	0.097
21	ND	0.155	0.126	0.141
24	ND	0.142	0.147	0.144
28	ND	0.240	0.121	0.181
30	ND	0.115	0.114	0.115

Note: “*” abnormal, eliminated; “ND” mean not detected

 

Measured Concentration in Water during the Test (Main Test)

Time (d)	Control	200 mg/kg
7	ND	ND
11	ND	ND
14	ND	ND
21	ND	ND
24	ND	ND
28	ND	ND
30	ND	ND

Note: “ND” means not detected

 

Lipid Content of Fish during the Test (Main Test)

Time (d)	Control	200 mg/kg
0	8.6%	8.1%
24	/	10.4%
28	/	10.5%
30	/	10.7%
24d-30d	NA	105%

Note: “NA” means not applicable; “/” not measured

 

Lipid Content in Diet (Main Test)

Time (d)	Lipid content
	1	2	Mean
0	13.24%	13.19%	13.2%
30	12.91%	13.58%	13.2%

 

Steady-State Biomagnification Factor (BM_Fss/BM_FssL)

Parameter	Calculation formula/source	200 mg/kg
Cfood(mg/kg)	Geometric mean of the measured concentration of the sample diet	206.1
Cfish(mg/kg)	Geometric mean of the measured concentration of the sample fish (Up24-30d)	0.144
T (d)	Duration of absorption phase	30
Lfish(%)	Average lipid content of fish (Up24-30d)	10.5
Lfood(%)	Average lipid content of diet (Up24-30d)	13.2
Lc	Lc=Lfish/Lfood	0.795
Steady-State Biomagnification factor (BMFss)	BMFss-Cfish/Cfood	6.98E-4
Lipid-Corrected Steady-State Biomagnification factor (BMFssL)	BMFssL=BMFss/Lc	8.789E-4

 

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the steady-state biomagnification factor (BMFss) of test item to rare minnow was 6.987E-4 and the lipid-corrected steady-state biomagnification factor (BMFssL) was 8. 789E-4. Given the extremely low BMF, the depuration step was not undertaken.

Executive summary:

Test method:

The purpose of the study was to determine the bioaccumulation potential of the test item, MLA-3202, in the rare minnow (Gobiocypris rarus) by dietary exposure. This study design is appropriate when aqueous exposure is not practical due to the test substance being insoluble and unable to maintain measurable concentrations in water.

The test concentration of 200 mg/kg MLA-3202 was selected based on the results of the range finding test. The fish in the control group were exposed to an untreated diet. The test exposure system was a flow-through exposure system. Each group consisted of a 20 L fish tank which contained 50 fish. There were three tanks in the test group and one tank in the control. The test was conducted with a 30 day uptake phase and no depuration phase based on the finding from the range-finder test that uptake of MLA-3202 in the fish is insignificant.

During the uptake phase, the fish were fed and observed daily to evaluate the number of mortalities and the number of individuals exhibiting signs of abnormal behavior. The water quality was tested once a week. During the test, the control and the test diets were made each week. Samples of water, fish and diet were collected to measure the concentration of test item. Lipid contents in fish and diet were detected. Fish were randomly selected from each group to measure body length and body weight at the beginning and the end of the test to calculate the growth rate. The steady-state biomagnification factor (BMFss) and the lipid-corrected steady-state biomagnification factor (BMFssL) were calculated after the test.

 

Test results:

During the test, no death or other abnormal behaviors were observed in the control and test group.

Water quality: Total hardness and total organic carbon (TOC) of the test water were measured as 190 mg CaCQ3/L and 1.02 mg/L at the start of the test. The particulate matter was measured quarterly, with the value of 0.6±0.2mg/L. During the test, temperature was maintained at 22.2 - 23.8 °C, pH value was in a range of 7.64 - 7.96; and the dissolved oxygen concentration was in a range of 99.0% - 100.6% ASV.

For the nominal concentration of 200 mg/kg in the diet, the result of accuracy showed that the measured concentration was 91.5%-119% of the nominal, in the range of 80% - 120%. The result of homogeneity showed that the relative standard deviations (RSDs) in five batches of diets were 0.57% - 2.6%, less than 15%. The result of stability showed that the measured concentration of the diets was 81 . 5%-109% of the initial concentration, less than 20%. All the results above showed that the test item in the diets was well prepared and stable during the period of storage. The concentration during the uptake phase was calculated as 206.1 mg/kg by the geometric mean.

For the control group, the concentration of the test item in diets, fish and water were all lower than the LOQ. For the test group, the measured concentration in fish was 0.092 mg/kg - 0 .181 mg/kg, and in water it was lower than the LOQ.

Considering that the lipid content in fish from 24 days to 30 day was determined to be 10.5% , which was relatively stable, according to the mean measured concentration in fish and the mean lipid content from 24d to 30d, the BMFss value was calculated as 6.987E-4, the BMFssL value was 8. 789E-4.

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Qualifier:

no guideline followed

Principles of method if other than guideline:

Recognised and accepted calculation method: EPISuite BCFBAF Program (Version 3.01)

GLP compliance:

no

Details on estimation of bioconcentration:

EPISuite BCFBAF provides a bioconcentration factor (BCF) and bioaccumulation factor (BAF) prediction based on different regression equations or fixed values, selected on the basis of an initial classification between ionic and non-ionic compounds, and on the value of the EPISuite KOWWIN Program predicted Log KOW.

Key result

Type:

BCF

Value:

> 1.9 - < 2.27 dimensionless

Validity criteria fulfilled:

not applicable

Conclusions:

The EPISuite BCFBAF Program predicted the BCF/BAF values for four representative components of the chemical. The model predicted similar Log KOW values as the measured Log KOW values which increases the reliability of the predicted BCF/BAF values to a moderate to high reliability.
The EPISuite BCFBAF Program predicted Log BCF values of less than 2.7 for all four representative components indicating the substance is non-bioaccumulative with predicted Log BAF values of 1.90 to 2.27.

Executive summary:

The EPISuite BCFBAF Program predicted the BCF/BAF values for four representative components of the chemical. The model predicted similar Log KOW values as the measured Log KOW values which increases the reliability of the predicted BCF/BAF values to a moderate to high reliability.

The EPISuite BCFBAF Program predicted Log BCF values of less than 2.7 for all four representative components indicating the substance is non-bioaccumulative with predicted Log BAF values of 1.90 to 2.27.

Description of key information

The EPISuite BCFBAF Program predicted the BCF/BAF values for four representative components of the chemical. The model predicted similar Log KOW values as the measured Log KOWvalues which increases the reliability of the predicted BCF/BAF values to a moderate to high reliability.

The EPISuite BCFBAF Program predicted Log BCF values of less than 2.7 for all four representative components indicating the substance is non-bioaccumulative with predicted Log BAF values of 1.90 to 2.27.

In a specific OECD 305 dietary study, the BMFss value was calculated as 6.987E-4, the BMFssL value was 8. 789E-4. The substance has an extremely low bioaccumulation potential

Key value for chemical safety assessment

BMF in fish (dimensionless):

0.001

Additional information

QSAR:

As the notified substance is a UVCB, its BCF/BAF was predicted from the chemical structures of its four most major components: C18:1 (oleic); C16:0 (palmitic); C18:2 (linoleic); and C18:0 (stearic), which together comprise approximately 73.6% of the overall UVCB substance. While the reliability of the predicted Log KOWvalues is low, the predicted Log KOWvalues are within range of the experimentally measured values of 5.3 to >7.2; suggesting the overall BCF/BAF predictions have a moderate to high reliability.

Test method:

The purpose of the study was to determine the bioaccumulation potential of the test item, MLA-3202, in the rare minnow (Gobiocypris rarus) by dietary exposure. This study design is appropriate when aqueous exposure is not practical due to the test substance being insoluble and unable to maintain measurable concentrations in water.

The test concentration of 200 mg/kg MLA-3202 was selected based on the results of the range finding test. The fish in the control group were exposed to an untreated diet. The test exposure system was a flow-through exposure system. Each group consisted of a 20 L fish tank which contained 50 fish. There were three tanks in the test group and one tank in the control. The test was conducted with a 30 day uptake phase and no depuration phase based on the finding from the range-finder test that uptake of MLA-3202 in the fish is insignificant.

During the uptake phase, the fish were fed and observed daily to evaluate the number of mortalities and the number of individuals exhibiting signs of abnormal behavior. The water quality was tested once a week. During the test, the control and the test diets were made each week. Samples of water, fish and diet were collected to measure the concentration of test item. Lipid contents in fish and diet were detected. Fish were randomly selected from each group to measure body length and body weight at the beginning and the end of the test to calculate the growth rate. The steady-state biomagnification factor (BMFss) and the lipid-corrected steady-state biomagnification factor (BMFssL) were calculated after the test.

 

Test results:

During the test, no death or other abnormal behaviors were observed in the control and test group.

For the nominal concentration of 200 mg/kg in the diet, the result of accuracy showed that the measured concentration was 91.5%-119% of the nominal, in the range of 80% - 120%. The result of homogeneity showed that the relative standard deviations (RSDs) in five batches of diets were 0.57% - 2.6%, less than 15%. The result of stability showed that the measured concentration of the diets was 81 . 5%-109% of the initial concentration, less than 20%. All the results above showed that the test item in the diets was well prepared and stable during the period of storage. The concentration during the uptake phase was calculated as 206.1 mg/kg by the geometric mean.

For the control group, the concentration of the test item in diets, fish and water were all lower than the LOQ. For the test group, the measured concentration in fish was 0.092 mg/kg - 0 .181 mg/kg, and in water it was lower than the LOQ.

Considering that the lipid content in fish from 24 days to 30 day was determined to be 10.5% , which was relatively stable, according to the mean measured concentration in fish and the mean lipid content from 24d to 30d, the BMFss value was calculated as 6.987E-4, the BMFssL value was 8. 789E-4. Given the extremely low BMF, the depuration step was not undertaken.