(Z)-N-octadecyldocos-13-enamide

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:

28 oct 2019 - 14 Feb 2020

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)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Limit test conducted at a concentration of 100% v/v saturated solution.

- Sampling method:
Water samples were taken from the control and test group from the freshly prepared bulk test preparation on Days 0, 6, 13, 19, 27 and 32 and from the old or expired media on Days 1, 7, 14, 20, 28 and 33 (from a single replicate of each treatment group, changing systematically amongst replicates throughout the test) for quantitative analysis.
Sample bottles (From Day 13 onwards) were fortified by the addition of 2 mL of acetonitrile in order to stabilize the test item.

- Sample storage conditions before analysis:
The samples were sent for analysis on the same day as sampling

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Pre-experiments were performed using different test substance concentrations and solvents. The results obtained showed that no significant effect was noted in terms of increasing the test item concentration, however, grinding the test item appeared to improve the consistency of the achieved concentrations. Therefore the method described below was chosen.
- Method: On Days -2, -1, 0 and 2 to 30 a nominal amount of test item (220 mg), pre-ground using a pestle and mortar for approximately 1 minute, was dispersed in test water and the volume adjusted to 22 liters with the aid of propeller stirring at approximately 1500 rpm for approximately 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a Whatman 0.2 µm Polycap 36AS filter (first approximate 2 liters used to pre-condition the filter was discarded).
- Eluate: test water
- Controls: The control group was maintained under identical conditions but not exposed to the test item.
- Test concentration separation factor: Limit test conducted at a concentration of 100% v/v saturated solution.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Strain: not reported
- Source: Adult fathead minnows that produced the eggs for the test were bred at Covance CRS
Research Limited

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): - Method of collection of fertilised eggs: Each breeding tank was supplied with inverted plastic guttering for the fish to lay eggs on and be fertilized. Fertilized eggs were collected from the breeding tanks on 09 January 2020 and used for the definitive test. The eggs were visually inspected before introduction into the test system and were identified as being at early blastodisc stage.
- Subsequent handling of eggs: not reported

POST-HATCH FEEDING
- Start date: Start after larvae stage
- Type/source of feed: Day 7 - Day 11: less than 24-hour old special grade brine shrimp nauplii
Day 12 - Day 32: less than 24-hour old basic grade brine shrimp nauplii
- Amount given: Not reported
- Frequency of feeding: Not reported

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

33 d

Remarks on exposure duration:

28 days post-hatch period

Hardness:

152 - 164 mg CaCO3/L
9.19 °dH

Test temperature:

24 - 26 °C

pH:

7.46

Dissolved oxygen:

≥ 5.1 mg/L (equivalent to 60% ASV)

Conductivity:

513 µS/cm at 20 °C

Nominal and measured concentrations:

Nominal concentration:10 mg/L
Geometric mean measured concentration: 0.021 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 1 liter glass vessels were used from Day 0 to Day 14 and from Day 15 to the end of the test 5 liter glass vessels were used. The approximate volume of test preparation in each vessel was 400 mL from Day 0 to Day 5, 800 mL from Day 6 to Day 14 and 4000 mL from Day 15 to Day 33.
- Type: open
- Aeration: Via narrow bore glass tubes from Day 12 onwards
- Renewal rate of test solution (frequency/flow rate): Daily renewal of the test solutions, with the exception of Day 3 where there was no water change to avoid causing premature hatching of the eggs.
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature
- Total organic carbon: not reported
- Particulate matter: not reported
- Metals: Aluminium < 6 µg/L
Iron < 35 µg/L
Manganese = 1.5 µg/L
Sodium = 46.4 mg/L
Copper = 0.0764 mg/L
Lead < 1.5 µg/L
Nickel = 4.6 µg/L
- Pesticides: < 0.004 µg/L
- Chlorine: 26.28 mg/L
- Alkalinity: not reported
- Ca/mg ratio: not reported
- Salinity: not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: not reported

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods
- Light intensity:594 - 728 lux

EFFECT PARAMETERS MEASURED: The number of dead eggs (up to completion of hatching), dead and live larvae and sub-lethal effects of exposure were recorded daily. The criteria of death for eggs were marked loss of translucency and change in coloration leading to a white opaque appearance. The criteria of death for larvae and juvenile fish were one or more of the following: immobility, absence of respiratory movement, absence of heart beat, white opaque coloration and lack of reaction to mechanical stimulus. At the end of the test the length and wet weight of each surviving fish was determined.

VEHICLE CONTROL PERFORMED: No, as no solvent was used.

RANGE-FINDING STUDY
Due to the low solubility and achievable test concentration in the test media it was considered unnecessary to perform a preliminary range-finding test.

POST-HATCH DETAILS
- Begin of post-hatch period: Day 6
- No. of hatched eggs (alevins)/treatment released to the test chamber: 20

FERTILIZATION SUCCESS STUDY
Not performed.

Reference substance (positive control):

no

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.021 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Post-hatch Survival

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.021 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Remarks:

Body length

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.021 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

Body weight

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.021 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Details on results:

- Mortality/survival at embryo, larval, juvenile, and adult stages: There were no significant mortalities or sub-lethal effects of exposure observed in any of the test concentrations. Details can be found in ‘Any other information on results incl. tables’.
- Days to hatch or time to release of young: The start of egg hatching was observed on Day 4 of the test and completion of hatching was observed on Day 5 of the test.
- Numbers hatched: In control and treatment group all eggs successfully produced hatchlings.
- Number of fish in swim-up stage at one or more time periods (e.g., day x1, x2): not reported
- Observations on body length and weight of young and/or exposed parents at one or more time periods:
Mean body length: Control = 21.18 mm; Treatment = 21.04 mm
Mean wet weight: Control = 85.2 mg; Treatment = 83.4 mg
Details can be found in ‘Any other information on results incl. tables’.
- Number of healthy fish at end of test: Control = 75; Treatment = 71
- Type of and number with morphological abnormalities: Abnormal observations were observed in three replicates in the treatment group. The observations recorded were pale fish and bent spine; however, the numbers of fish recorded with these observations were very low. Therefore, these observations are considered to be due to defects occurring in a natural population, and not necessarily attributable to exposure to the test item. There were no sub-lethal effects observed in the control group throughout the test
- Type of and number with behavioural abnormalities: Not reported
- Effect concentrations exceeding solubility of substance in test medium: No, as a saturated solution in test medium was used.
- Analytical verification: Analysis of the fresh test preparations on Days 0, 6, 13, 19, 27 and 32 showed measured test concentrations to range from less than the Limit of Detection (ND) of the analytical method, determined to be 0.01 µg/L or less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.02 µg/L to 0.060 µg/L. Measured test concentration of the old test preparations on Days 1, 7, 14, 20, 28 and 33 was observed to be between less than the Limit of Detection of the analytical method and 0.042 µg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data. The geometric mean measured concentration for the 100% v/v saturated solution was determined to be 0.021 µg/L. Duplicate samples were sent for analysis at the following time points due to anomalous results being generated: Day 19 (fresh) and Day 20 (old) as the test samples did not show any test item present, analysis of the duplicate samples confirmed the result for Day 19 but showed test item present in the Day 20 sample (0.02 µg/L) confirming that the test system had been dosed correctly. Day 27 (fresh) as the test sample was shown to be less than the LOQ, the Day 28 (old) sample showed that the test system had been dosed with test item as a value of 0.050 µg/L was achieved, however, the analytical control samples failed and as such this value could not be used. Subsequent analysis of the duplicate samples showed that no test item was present. The original samples for Day 32 (fresh) and Day 33 (old) were rejected due to variability in results generated across the batch. The duplicate analysis gave results of 0.060 at Day 32 confirming that the test system was dosed correctly and none detected at Day 33. Due to the extremely low water solubility (practically insoluble) of the test substance it was not surprising to have values generated which were very close to or below the limit of quantification or detection of the analytical method. This was apparent on some occasions for the fresh media samples sent for analysis, however the aged samples subsequently showed a higher concentration within the test media. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ and highlights the issues and problems of testing test items that are practically insoluble in the test media.

Results with reference substance (positive control):

No reference substance used.

Reported statistics and error estimates:

Geometric Mean Measured Test Concentrations
The geometric mean measured test concentrations of the samples were calculated by taking the square root of the product of the measured concentration at the start of the media renewal period and the measured concentration at the end of the media renewal period.

Lowest Observed Effect Concentration and No Observed Effect Concentration Determinations
As the study was conducted as a Limit Test, the LOEC could not be determined. For the estimation of the NOEC the hatching and post-hatch survival data for the control and test group were compared using Fisher’s Exact Binomial Test. The length and wet weight data for the control and each test group obtained on termination of the test were compared using the Student t-test for Homogeneous Variances incorporating Levene’s Test on Variance Homogeneity and Shapiro-Wilk’s Test on Normal Distribution. All results were calculated using the ToxRat Professional computer software package (ToxRat).

Table 1: Number of Dead Eggs

Geometric Mean Measured Concentration (µg/L)	Number of Dead Eggs (Initial Population = 20)
	Replicate	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6*	Total
Control	1	0	0	0	0	0	0	0
	2	0	0	0	0	0	0	0
	3	0	0	0	0	0	0	0
	4	0	0	0	0	0	0	0
0.021	1	0	0	0	0	0	0	0
	2	0	0	0	0	0	0	0
	3	0	0	0	0	0	0	0
	4	0	0	0	0	0	0	0

* All surviving eggs hatched by Day 6

 

Table 2: Number of Dead Larvae/Fish (Day 1 - Day 17)

Geometric Mean Measured Concentration (µg/L)	Number of Dead Fish (Non-cumulative)
	Replicate	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7	Day 8	Day 9	Day 10	Day 11	Day 12	Day 13	Day 14	Day 15	Day 16	Day 17
Control	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2	0	0	0	0	0	0	0	0	0	0	0	2	0	0	0	0	0
	3	0	0	0	0	0	0	0	0	0	0	0	2	0	0	0	0	0
	4	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0
0.021	1	0	0	0	0	0	0	0	1	0	1	0	0	0	0	0	0	0
	2	0	0	0	0	0	0	0	2	1	0	1	0	0	0	0	0	0
	3	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0
	4	0	0	0	0	0	0	0	0	2	0	0	0	0	0	0	0	0

 

Table 3: Number of Dead Larvae/Fish (Day 18 - Day 33)

Geometric Mean Measured Concentration (µg/L)	Number of Dead Fish (Non-cumulative)
	Replicate	Day 18	Day 19	Day 20	Day 21	Day 22	Day 23	Day 24	Day 25	Day 26	Day 27	Day 28	Day 29	Day 30	Day 31	Day 32	Day 33
Control	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2	0	0	0	0	0	0	0	0	0	0	0	2	0	0	0	0
	3	0	0	0	0	0	0	0	0	0	0	0	2	0	0	0	0
	4	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0
0.021	1	0	0	0	0	0	0	0	1	0	1	0	0	0	0	0	0
	2	0	0	0	0	0	0	0	2	1	0	1	0	0	0	0	0
	3	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0
	4	0	0	0	0	0	0	0	0	2	0	0	0	0	0	0	0

 

Table 4: Hatching and Post Hatch Survival

Geometric Mean Measured Concentration (µg/L)	Replicate	Hatching Rate	Mean Hatching Rate	Survival Rate	Mean Survival Rate
		%	%	%	%
Control	1	100	100	100	94
	2	100		90
	3	100		90
	4	100		95
0.021	1	100	100	90	89
	2	100		80
	3	100		95
	4	100		90

 

Table 5: Fish Length and Weight Data - Control

Fish Length and Weight Data - Control
Replicate	1		2		3		4
Fish	Length (mm)	Wet Weight (mg)	Length (mm)	Wet Weight (mg)	Length (mm)	Wet Weight (mg)	Length (mm)	Wet Weight (mg)
1	21.79	98.9	21.92	89.6	22.44	109.3	19.95	83.2
2	19.35	61.2	22.98	103.2	20.48	83.5	21.47	89.3
3	22.06	82.1	22.73	98.7	20.42	73.9	19.63	66.8
4	21.02	74.1	22.01	81.7	21.51	86.2	22.68	124.7
5	20.91	78.8	23.59	116.3	19.4	64.8	23.43	136.1
6	21.16	84.2	22.05	85.8	21.27	82.8	21.83	96.6
7	21.79	82.8	22.31	99.4	22.28	83.2	20.58	73.7
8	21.21	86.1	19.52	64.6	19.07	64.9	22.18	87.9
9	19.36	62.7	23.66	98.7	20.54	94	20.06	68.2
10	20.31	69.7	20.8	79.4	22.39	87.4	23.14	105.1
11	20.3	76.6	21.6	76.7	20.84	95	22.39	94.8
12	20.61	68.3	22.75	113.3	19.98	79.9	22.35	88.3
13	23.33	115.6	20.14	81.5	20.45	87.4	20.82	69.4
14	21.02	91.2	17.69	54.4	21.36	101.5	21.52	91.9
15	19.82	68.5	21.4	75.4	22.47	96.1	17.72	53.8
16	22.56	100.4	23.19	101	21.6	90.1	20.61	98.4
17	21.8	94.6	21.83	82.5	20.75	85.2	19.54	86.8
18	18.92	52.7	18.17	45.4	21.55	89.4	20.88	82.5
19	22.17	84.9	-	-	-	-	20.6	82.4
20	20.15	66	-	-	-	-	-	-
Average	20.98	80	21.57	86	21.04	86.4	21.13	88.4

 

Table 6: Fish Length and Weight Data - Treatment

Fish Length and Weight Data 0.021 µg/L (Geometric Mean Measured Concentration)
Replicate	1		2		3		4
Fish	Length (mm)	Wet Weight (mg)	Length (mm)	Wet Weight (mg)	Length (mm)	Wet Weight (mg)	Length (mm)	Wet Weight (mg)
1	21.44	77.6	20.97	79.8	19.74	69.5	22.91	113.7
2	22.44	101.7	21.37	88.4	20.3	79	19.97	73.7
3	19.98	64.6	22.26	90.8	20.51	83.8	21.31	116.3
4	20.11	125.1	20.38	66.8	21.65	98.2	22.54	100.1
5	20.35	69.4	21.87	94.7	21.85	82.1	21.95	90.8
6	20.48	75.8	22.41	87.5	20.21	70.8	21.65	91.2
7	22.88	94.9	21.53	85.8	20.66	69.3	19.78	66.9
8	21.48	100.9	21.45	69.3	19.67	78.9	18.06	45
9	22.5	95.1	21.23	80.4	20.78	86.2	22.41	109
10	22.22	104.2	20.91	87.7	20.92	80.3	20.86	73
11	22.35	99.3	22.94	100.5	20.08	72.5	20.76	78.3
12	20.84	74.1	20.93	92.1	20.01	72.7	21.28	87.7
13	21.49	92.3	21.15	80	19.57	64.7	19.43	65.6
14	21.82	78.3	23.17	115.8	22.52	100.4	19.36	68.5
15	19.78	67.7	19.63	63	21.07	79.9	21.06	91.8
16	21.73	97.6	21.36	90.9	21.16	83	20.56	67.4
17	20.27	62.4	-	-	21.02	63	18.55	63.3
18	20.29	63.9	-	-	20.91	80.9	21.12	101.6
19	-	-	-	-	20.64	75.9	-	-
20	-	-	-	-	-	-	-	-
Average	21.25	85.8	21.47	85.8	20.7	78.5	20.75	83.6

 

Table 7: Number of Larvae with Abnormal Observations (Day 1 - Day 17)

Geometric Mean Measured Concentration (µg/L)	Number of Larvae with Abnormal Observations
	Replicate	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7	Day 8	Day 9	Day 10	Day 11	Day 12	Day 13	Day 14	Day 15	Day 16	Day 17
Control	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	3	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	4	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
0.021	1	0	0	0	0	0	0	0	1B	1B	0	0	0	0	0	0	0	0
	2	0	0	0	0	1B	1B	1B 2P	2P	1P	1P	0	0	0	0	0	0	0
	3	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	4	0	0	0	0	0	0	3P	1B 2P	1P	1P	0	0	0	0	0	0	0

 

 

Table 8: Number of Larvae with Abnormal Observations (Day 18 - Day 33)

Geometric Mean Measured Concentration (µg/L)	Number of Larvae with Abnormal Observations
	Replicate	Day 18	Day 19	Day 20	Day 21	Day 22	Day 23	Day 24	Day 25	Day 26	Day 27	Day 28	Day 29	Day 30	Day 31	Day 32	Day 33
Control	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	3	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	4	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
0.021	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	3	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	4	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

Table 9 Validity criteria for OECD 210

Criterion from the guideline	Outcome	Validity criterion fulfilled
The dissolved oxygen concentration should be >60% of the air saturation value throughout the test.	>60%	Yes
The water temperature should not differ by more than ± 1.5 °C between test chambers or between successive days at any time during the test, and should be within the temperature ranges specified for the test species (Annex 2).	23.7 - 25.7 °C	Yes
The analytical measure of the test concentrations is compulsory.	A detailed and considered analytical verification of the treatment concentrations was performed	Yes
Overall survival of fertilised eggs and post-hatch success in the controls and, where relevant, in the solvent controls should be greater than or equal to the limits defined in Annex 2.	Hatching success rate = 100 % Post-hatch survival = 94 %	Yes

 

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

No toxic effects at saturation in test medium.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Remarks:

No toxicity within the range of water-solubility

Effect concentration:

>= 0.002 µg/L

Additional information

A fish early life stage study (Covance, 2020) according to OECD 210 (2013) was performed under GLP conditions using Pimephales promelas. The test was initiated with newly fertilized eggs (<24 h post fertilization and before beginning of gastrulation). Due to the limited solubility of the test item and low achievable test concentrations in the test media a "limit test" was conducted at a concentration of 100% v/v saturated solution (nominal concentration of 10 mg/L loading rate) to confirm that at the highest attainable test concentration no adverse reactions to exposure were observed.

Newly fertilized fathead minnow eggs (four replicates of 20 eggs per group) were exposed to a solution of the test item for a period of 33 days under semi-static test conditions (daily renewal).

Water samples were taken from the control and test group from the freshly prepared bulk test preparation on Days 0, 6, 13, 19, 27 and 32 and from the old or expired media on Days 1, 7, 14, 20, 28 and 33.

Analysis of the fresh test preparations on Days 0, 6, 13, 19, 27 and 32 showed measured test concentrations to range from less than the Limit of Detection (ND) of the analytical method, determined to be 0.01 µg/L or less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.02 µg/L to 0.060 µ/L.

Measured test concentration of the old test preparations on Days 1, 7, 14, 20, 28 and 33 was observed to be between less than the Limit of Detection of the analytical method and 0.042 µg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data. The geometric mean measured concentration for the 100% v/v saturated solution was determined to be 0.021 µg/L. Due to the extremely low water solubility (practically insoluble) of the test substance it was not surprising to have values generated which were very close to or below the limit of quantification or detection of the analytical method. This was apparent on some occasions for the fresh media samples sent for analysis, however the aged samples subsequently showed a higher concentration within the test media. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ and highlights the issues and problems of testing test items that are practically insoluble in the test media. Over the duration of the test there were no significant mortalities or sub-lethal effects of exposure resulting from the exposure of fish larvae to a test concentration of 0.021 µg/L. The application of the test material to newly laid eggs of fathead minnows was considered to have no effect on the survival or growth of the larvae. The No Observed Effect Concentration (NOEC) was >= 0.021 µg/L. The Lowest Observed Effect Concentration (LOEC) could not be determined as the study was conducted as a Limit Test and no significant effects were apparent at the dose level tested.