C16-18-(even numbered)-alkyl fatty acid, compound with C16-18-(even numbered)-alkylamine

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

Media preparation
The test medium used was ASTM medium. It was reconstituted with chemical reagents and deionised water. The test medium was performed several times a week.

Test solution preparation
Since the test item is poorly soluble under the test conditions, a saturated solution of test item (limit of solubility LS) was prepared. A solution with a loading rate of approximately 1g/L was prepared and stirred with an agitator for several days. Then, this stock solution was filtered with cellulose acetate filters at 0.45 µm.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Fertilised eggs of Danio rerio produced from the laboratory genitors were used as soon as possible after fertilisation. At the laboratory, male and female genitors were separated in aquariums with a continuous renewal of the water.
The day before the experiment, male and females were added in the nest box. Egg laying and fertilisation occurred at « dawn », simulated with lighting. Eggs were harvested and a selection was done under microscope in order to select fertilised eggs according to Annex 3 of OECD 236 guideline.
Breeding conditions for genitors were the following:
Medium: water from the water system (mix of tapwater and deionized water)
Temperature: 23 °C ± 2
Light/dark cycle: 16 h/8 h
Dissolved oxygen concentration: = 80 % of the air saturation value
Feeding: twice a day ad libidum (once with small granular and once with freezing food (chironomus, artemia)

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

34 d

Post exposure observation period:

Exposure took place on embryo as well as for 30 days post-hatch.

Test temperature:

26.1 - 27.1°C (min - max)

pH:

7.2 - 7.8 (min-max)

Dissolved oxygen:

69.8 - 103.0% (min-max)

Nominal and measured concentrations:

Nominal: Five concentrations at LS/81 – LS/27 – LS/9 – LS/3 and LS were prepared in ASTM medium. A control was set up with the test medium only. LS = limit of water solubility (i.e. saturated solu33tion)
Measured: see table 1.

Details on test conditions:

Test containers
The test was performed in glass crystallizer of 50 mL, filled to 25 mL during the five first days, in glass crystallizer of 400 mL filled to 125 mL between days 5 and days 14, in glass aquarium of 1.5L filled to 250 mL between days 14 and days 21 and filled to 500 mL until the end of the test. Fish loading rate was less than 5 g.L-1. The maximal fish weight at the end of the test was 0.2666g per aquarium for a volume of 500 mL.
The test was performed under semi-static conditions (renewal three times a week). The test organisms were retained in the test vessels with the exception of days of change of test containers whilst a proportion (at least two thirds) of the test solution / control volume was changed.

Performing the ecotoxicity test
Five concentrations at LS/81 – LS/27 – LS/9 – LS/3 and LS were prepared in ASTM medium. A control was set up with the test medium only. There were 4 replicates for each treatment with 20 eggs per replicate. The test was performed in an experimental room maintained at 26° C ± 1.5 with a day / night cycle of 16h / 8h. pH and oxygen of test solutions were recorded before and after renewal. Before hatching, the number of coagulated eggs was noted, and after hatching, the number of deaths in each replicate was recorded daily. The test was performed during 30 days post hatching. At the end of the test, fish were measured individually and then weighed in batches. Means of lengths and weights were calculated by replicate. Two days post hatching, fish were fed with Nobilfluid ® and live rotifera (Brachionus plicatilis) twice or three times a day. Six days post hatching, Nobilfluid ® was stopped and live artemia (Artemia salina) were added.

Euthanasia
Fish were euthanized in order to carry out the measurements of weight and length. For this, fish were placed for about two minutes in a crystallizer containing sodium methanesulfonate or MS222 (CAS number 886-86-2) at a lethal concentration of 300 mg/L.

Reference substance (positive control):

no

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

81.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

34 d

Dose descriptor:

EC10

Effect conc.:

5.75 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

6.67 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

dry weight

Details on results:

See Table 1 to 15.

Reported statistics and error estimates:

The hatching, mortality and abnormality percentage were determined for each concentration. EC10 (Effect Concentration for 10% effect), EC50 and confidence limits were obtained by calculation according to Regtox® Macro (“Vindimian”), using the Hill model.
The equation of Hill (1910) was described to model the binding of oxygen to haemoglobin. It was shown to be relevant for many other mechanisms like binding of a ligand to a receptor, Michaelian or non Michaelian enzymatic kinetics, dose or concentration response in toxicology and ecotoxicology (Vindimian 1983, Garric 1990). The logistic model is also used which is similar to the Hill model.
The model used in this macro is written in the form proposed by Duggleby in 1981. It fits different cases where the measurement of a biological parameter is plotted versus concentration or dose. Two parameters: Hill number and EC50 are characteristics of the probability function written as follows:

f(x) = 1 / [ 1 + (EC50 / x)^(nh)]

It should be noted that f(x)=1/2 when x = EC50.
The fitting is based on the algorithm of Marquardt (1963) which is robust and fast. It needs initial estimates of the parameters and optimize them by successive iterations.
The confidence intervals on the parameters are estimated by a bootstrap simulation which is entirely non parametric and is considered to be well adapted to non linear models (Efron 1990).
The NOEC (No Observed Effect Concentration) were determined by statistical test from replicate data on hatching, mortality and on length and weight. Statistical calculations were made using R statistical software.

 Table 1: Test concentrations expressed as geometrical means

	Test item geometrical mean (µg/L)
	After hatching	End of the test
LS/81	5.11	4.38
LS/27	9.41	6.67
LS/9	22.07	12.13
LS/3	81.20	34.12
LS	651.79	651.79
Control	<LoD	< LoD

 

 

 

Table 2: Hatching and survival rates.

	Replicate	Number of hatched larvae on D5	Hatching rate per replicate (%)	Mean hatching rate (%)	Number of survivors at the end of the test	Survival rate per replicate (%)	Mean survival rate (%)
Control	R1	19	95.00	93.8	15	78.9	76.0
	R2	20	100.00		15	75.0
	R3	17	85.00		13	76.5
	R4	19	95.00		14	73.7
LS/81	R1	20	100.00	97.5	15	75.0	75.7
	R2	18	90.00		14	77.8
	R3	20	100.00		14	70.0
	R4	20	100.00		16	80.0
LS/27	R1	17	85.00	93.8	4	23.5	21.6
	R2	20	100.00		3	15.0
	R3	18	90.00		5	27.8
	R4	20	100.00		4	20.0
LS/9	R1	20	100.00	96.3	2	10.0	3.9
	R2	19	95.00		0	0.0
	R3	20	100.00		0	0.0
	R4	18	90.00		1	5.6
LS/3	R1	20	100.00	100.0	0	0.0	0.0
	R2	20	100.00		0	0.0
	R3	20	100.00		0	0.0
	R4	20	100.00		0	0.0
LS	R1	1	5.00	1.3	0	0	0
	R2	0	0.00		-	0
	R3	0	0.00		-	0
	R4	0	0.00		-	0

 

Table 3: Weight and length of surviving larvae at the end of the test

	Replicate	Length (mm)	Average length (mm)	Wet weight (g)	Average wet weight (g)	Dry weight (g)	Average dry weight (g)
C01	R1	12.96	13.31	17.77	17.25	3.15	3.20
	R2	13.27		14.94		2.99
	R3	13.55		17.55		3.60
	R4	13.45		18.75		3.06
C02	R1	13.34	13.08	17.77	16.54	3.24	3.29
	R2	13.28		17.57		3.71
	R3	12.60		14.86		3.24
	R4	13.10		15.96		2.97
C03	R1	10.51	10.51	10.48	10.13	2.18	1.98
	R2	12.03		14.20		2.97
	R3	10.85		11.68		2.20
	R4	8.64		4.15		0.57
C04	R1	9.16	9.74	5.20	6.10	0.40	0.45
	R4	10.32		7.00		0.50

 

Table 4: Number of dead eggs per day and cumulative hatch per replicate in the control group

	Control group
Date	R1		R2		R3		R4
	Dead	Hatched	Dead	Hatched	Dead	Hatched	Dead	Hatched
D1	0	0	0	0	1	0	0	0
D2	0	0	0	0	0	0	0	0
D3	0	2	0	5	0	3	0	4
D4	0	19	0	20	0	17	0	19
D5	1	19	0	20	2	17	1	19

 

Table 5: Number of dead eggs per day and cumulative hatch per replicate in the LS/81 group

	LS/81
Date	R1		R2		R3		R4
	Dead	Hatched	Dead	Hatched	Dead	Hatched	Dead	Hatched
D1	0	0	0	0	0	0	0	20
D2	0	0	0	0	0	0	0	20
D3	0	5	0	3	0	6	0	20
D4	0	20	0	18	0	20	0	20
D5	0	20	1	18	0	20	0	20

 

Table 6: Number of dead eggs per day and cumulative hatch per replicate in the LS/27 group

	LS/27
Date	R1		R2		R3		R4
	Dead	Hatched	Dead	Hatched	Dead	Hatched	Dead	Hatched
D1	0	0	1	0	0	0	0	0
D2	0	0	0	0	0	0	0	0
D3	0	2	0	3	0	5	0	6
D4	0	17	0	20	0	18	0	20
D5	1	17	0	20	1	18	0	20

 

Table 7: Number of dead eggs per day and cumulative hatch per replicate in the LS9 group

	LS/9
Date	R1		R2		R3		R4
	Dead	Hatched	Dead	Hatched	Dead	Hatched	Dead	Hatched
D1	0	0	0	0	0	0	0	0
D2	0	0	0	0	0	0	0	0
D3	0	3	0	3	0	7	0	4
D4	0	20	0	18	0	19	0	18
D5	0	20	1	19	0	20	2	18

 

Table 8: Number of dead eggs per day and cumulative hatch per replicate in the LS/3 group

	LS/3
Date	R1		R2		R3		R4
	Dead	Hatched	Dead	Hatched	Dead	Hatched	Dead	Hatched
D1	0	0	0	0	0	0	0	0
D2	0	0	0	0	0	0	0	0
D3	0	5	0	4	0	2	0	3
D4	0	20	0	20	0	20	0	20
D5	0	20	0	20	0	20	0	20

 

Table 9: Number of dead eggs per day and cumulative hatch per replicate in the LS group

	LS
Date	R1		R2		R3		R4
	Dead	Hatched	Dead	Hatched	Dead	Hatched	Dead	Hatched
D1	0	0	0	0	0	0	0	0
D2	14	0	11	0	19	0	19	0
D3	3	0	1	0	0	0	0	0
D4	0	1	0	0	0	0	0	0
D5	2	1	6	0	1	0	1	0

 

Table 10: Number of dead larvae per day in the control group

	Control group
Date	R01	R02	R03	R04
	Dead	Dead	Dead	Dead
D4	0	0	0	0
D5	0	0	0	1
D6	0	0	0	0
D7	0	0	0	0
D8	0	0	0	0
D9	0	0	0	0
D10	0	0	0	0
D11	0	0	0	0
D12	0	0	0	0
D13	0	0	0	0
D14	3	2	1	3
D15	0	0	0	0
D16	0	0	0	0
D17	0	0	0	0
D18	0	3	2	0
D19	0	0	0	0
D20	0	0	0	0
D21	0	0	0	0
D22	0	0	0	0
D23	1	0	1	1
D24	0	0	0	0
D25	0	0	0	0
D26	0	0	0	0
D27	0	0	0	0
D28	0	0	0	0
D29	0	0	0	0
D30	0	0	0	0
D31	0	0	0	0
D32	0	0	0	0
D33	0	0	0	0
D34	0	0	0	0

 

Table 11: Number of dead larvae per day in the LS/81 group

	LS/81
Date	R01	R02	R03	R04
	Dead	Dead	Dead	Dead
D4	0	0	0	0
D5	0	0	0	0
D6	0	1	0	0
D7	0	0	0	0
D8	0	0	0	0
D9	0	0	0	0
D10	0	0	0	0
D11	0	0	0	0
D12	0	0	0	0
D13	0	0	0	0
D14	2	2	3	4
D15	0	0	0	0
D16	0	0	0	0
D17	0	0	0	0
D18	2	0	1	0
D19	0	0	0	0
D20	0	0	0	0
D21	0	0	0	0
D22	0	0	0	0
D23	1	1	2	0
D24	0	0	0	0
D25	0	0	0	0
D26	0	0	0	0
D27	0	0	0	0
D28	0	0	0	0
D29	0	0	0	0
D30	0	0	0	0
D31	0	0	0	0
D32	0	0	0	0
D33	0	0	0	0
D34	0	0	0	0

 

Table 12: Number of dead larvae per day in the LS/27 group

	LS/27
Date	R01	R02	R03	R04
	Dead	Dead	Dead	Dead
D4	0	0	0	0
D5	0	0	0	0
D6	0	0	2	1
D7	0	0	0	0
D8	0	0	0	0
D9	0	0	0	0
D10	0	0	0	0
D11	0	0	0	1
D12	0	0	0	0
D13	5	6	3	9
D14	5	7	2	1
D15	1	0	0	0
D16	0	0	0	0
D17	0	0	0	0
D18	0	0	3	3
D19	0	1	2	0
D20	0	0	0	0
D21	0	0	0	0
D22	0	0	0	0
D23	2	3	1	1
D24	0	0	0	0
D25	0	0	0	0
D26	0	0	0	0
D27	0	0	0	0
D28	0	0	0	0
D29	0	0	0	0
D30	0	0	0	0
D31	0	0	0	0
D32	0	0	0	0
D33	0	0	0	0
D34	0	0	0	0

 

Table 13: Number of dead larvae per day in the LS/9 group

	LS/9
Date	R01	R02	R03	R04
	Dead	Dead	Dead	Dead
D4	0	0	0	0
D5	0	0	0	0
D6	0	1	0	0
D7	0	0	0	0
D8	0	0	0	0
D9	0	0	0	0
D10	0	0	0	0
D11	3	0	0	3
D12	4	6	4	6
D13	8	8	1	5
D14	2	1	11	0
D15	0	0	0	0
D16	0	0	0	0
D17	0	0	0	0
D18	1	0	0	2
D19	0	1	1	0
D20	0	0	0	0
D21	0	0	0	0
D22	0	0	0	0
D23	0	2	2	1
D24	0		1	0
D25	0			0
D26	0			0
D27	0			0
D28	0			0
D29	0			0
D30	0			0
D31	0			0
D32	0			0
D33	0			0
D34	0			0

 

Table 14: Number of dead larvae per day in the LS/3 group

	LS/3
Date	R01	R02	R03	R04
	Dead	Dead	Dead	Dead
D4	0	0	0	1
D5	0	1	0	0
D6	0	2	0	0
D7	0	0	0	0
D8	6	0	0	0
D9	0	0	0	0
D10	3	0	0	1
D11	7	3	1	1
D12	3	7	6	11
D13	2	3	5	6
D14		3	5
D15		1	1
D16		0	0
D17		1	1
D18			1

 

Table 15: Number of dead larvae per day in the LS group

	LS
Date	R01	R02	R03	R04
	Dead	Dead	Dead	Dead
D3	1

 

Validity criteria fulfilled:

yes

Conclusions:

34d-EC10 was 5.75 µg/L for larval survival.

Executive summary:

Long-term toxicity of the registered substance to fish was evaluated in a GLP study according to OECD 210 TG. Embryo and larvae were exposed to graded concentrations of the test item for 34 days in quadruplicate of 20 eggs each under semi-static conditions (control ; limit of solubility LS ; LS/3 ; LS/9 ; LS/27 ; LS/81). Solutions were renewed 3 times a week. Actual concentrations were monitored on a regular basis. Only one larvae hatched at LS and died the following day. All larvae died in the LS/27 treatment. NOEC/EC10 were determined for hatching rate, survival rate and growth (dry weight). The lowest of the three was EC10 for survival rate, which was 5.75 µg/L.

Description of key information

Long-term toxicity of the registered substance to fish was evaluated in a GLP study according to OECD 210 TG. Embryo and larvae were exposed to graded concentrations of the test item for 34 days in quadruplicate of 20 eggs each under semi-static conditions (control ; limit of solubility LS ; LS/3 ; LS/9 ; LS/27 ; LS/81). Solutions were renewed 3 times a week. Actual concentrations were monitored on a regular basis. Only one larvae hatched at LS and died the following day. All larvae died in the LS/27 treatment. NOEC/EC10 were determined for hatching rate, survival rate and growth (dry weight). The lowest of the three was EC10 for survival rate, which was 5.75 µg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

5.75 µg/L