Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts

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:

30 March - 12 May 2022

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

Details on sampling:

PREPARATION OF SAMPLES
Samples Nominally ≤ 160 μg/L
An accurate volume of test solution was transferred, using glass pipettes into glass vessels and accurately fortified (if required). The solutions were then diluted, using glass pipettes into glass vessels, ×2 with 0.2% acetic acid in LC-MS methanol. The solutions are further diluted, at least ×2 with 0.1:50:50 acetic acid: water: methanol (v/v/v) and aliquot(s) taken for analysis by LC-MS/MS.
Excess sample dilutions were stored refrigerated.
Samples Nominally > 160 μg/L
An accurate volume of test solution was transferred, using glass pipettes into glass vessels and accurately fortified (if required). The solutions were then diluted, using glass pipettes into glass vessels, ×2 with 0.2% acetic acid in LC-MS methanol and further diluted (as required, minimum of ×2) with 0.1:50:50 acetic acid: water: methanol (v/v/v) to within the calibration range. Aliquot(s) of the diluted solution(s) were then taken for analysis by LC-MS/MS.
Excess sample dilutions were stored refrigerated.

Vehicle:

yes

Remarks:

reverse-osmosis water

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: This study was run with a dilution water control and nominal Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts concentrations of 0.043, 0.094, 0.207, 0.455 and 1.0 mg a.i./L.
Dosing stocks were prepared by dissolving a known amount of Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts (see table in Any other information on materials and methods below) in 2000 mL of reverse-osmosis (RO) water in a volumetric flask. The amount of test substance weighed into each dosing stock was adjusted, based on the test substance purity (39.0%), to provide the test dose in mg a.i./L. A solution of 2000 mL RO water was incorporated in the dilution water control mixing apparatus. All stocks were treated similarly, approximately 5 minutes stirring used to ensure the test substance had dissolved into solution.
- Controls: Dilution water
- Chemical name of vehicle: Reverse osmosis water
- Test concentration separation factor: Approx 2.2
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All test substance stocks were observed to be clear and colourless with bubbles at the surface of the solution.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Source: Broodstock adult fish held at Scymaris. The broodstock were held under similar conditions as those used during the study. The broodstock were held under artificial lighting and fed daily using a commercial fish food, frozen adult brine shrimp (Artemia spp) and bloodworm. Broodstock showed no evidence of disease during the two months prior to study.
- Newly fertilised (<24 hours old) eggs were used.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- 29 breeding groups (1 group: 4 males and 8 females) were used to provide the eggs used in the study
- Egg incubation cups (1 per replicate tank) made from 8 cm lengths of 5 cm diameter glass tubing with nylon mesh stuck to the bottom of each cup using silicone sealant were used. The cups were suspended in the test vessels and oscillated vertically approximately 20-50 mm at 2 oscillations per minute to maintain a non-turbulent water flow over the eggs while ensuring complete submergence of the eggs.

POST-HATCH FEEDING
The hatched larvae/fry were fed twice daily (except for the start and end of the exposure).
From hatch day until Day 7 post-hatch, the fry were fed on a powdered commercial fry food fed ad libitum per tank per feed.
Newly hatched brine shrimp Artemia (typically up to 60 mL of culture per tank per feed) were fed from Day 2 post-hatch.
A pelleted commercial food, fed ad libitum from Day 17 post-hatch, was also introduced into the diet.
Feeding was in excess, while not reducing water quality conditions, and approximately equal across replicates based on numbers of fry.
Representative batches of the food types used in this study were analysed for pesticides, PCBs and metals.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

33 d

Remarks on exposure duration:

Total exposure duration is from egg to 28 days post hatch

Hardness:

The water hardness measured in the dilution water control and nominal 1.0 mg a.i./L test solutions at test start were 67.3 and 67.7 mg/L CaCO3, respectively. The water hardness in the dilution water control and nominal 1.0 mg a.i./L test solutions at the end of the test were 87.7 and 88.3 mg/L CaCO3, respectively.

Test temperature:

The temperature of the test solutions measured on six occasions throughout the study ranged from 23.4 to 25.2°C. Temperature data are shown in Table 9 (attached).
The continuous temperature measurement, recorded in the dilution water control replicate D test vessel, ranged from 24.4 to 25.9°C. Weekly ranges are shown in Table 10 (attached).

pH:

The pH values of the test solutions, measured throughout the study ranged from 7.38 to 7.86. pH data are shown in Table 7 (attached). The overall mean pH value was 7.72.

Dissolved oxygen:

The dissolved oxygen (DO) concentrations of the test solutions, measured throughout the study ranged from 75.2 to 98.0% of the air saturation value (ASV) with data shown in Table 8 (attached). The overall mean DO concentration was 92.9% ASV.

Nominal and measured concentrations:

Nominal concentrations (based on registered substance): 0.043, 0.094, 0.207, 0.455 and 1.0 mg a.i./L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquaria
- Size of vessel: Approx. 9.5 litre working capacity
- Type: open
- Type of flow-through: peristaltic
- Renewal rate of test solution (frequency/flow rate): nominal rate of 100 mL/min to individual tanks; approximately 15 tank volume replacements per day to each of the 4 replicate test vessels
- No. of organisms per vessel: 20 embryos, <24 hours old
- No. of vessels per concentration (replicates): four replicate aquaria per treatment
- No. of vessels per vehicle control (replicates): 4 replicates
- Biomass loading rate: The fish stocking density did not exceed 0.5 g/L/day or 5 g/L at any time, this was confirmed using wet weight data at test end (see additional information on results).

A test rig schematic is shown in Figure 1 (attached)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was town’s water, which had been passed through activated carbon and dechlorinated using a sodium thiosulphate solution. Salts were added, as required, to maintain minimum hardness levels, and the treated water passed through an ultraviolet steriliser and filtered to 10 μm. The supply was then delivered to a temperature-controlled tank adjacent to the test laboratory which was set to the nominal test temperature of 25 ± 1.5°C. It was finally re-filtered to 1 μm, in the laboratory, before distribution to the test rigs.
The dechlorinated water is regularly monitored for routine parameters (e.g. pH, hardness, conductivity, alkalinity). In addition, the dechlorinated supply is periodically monitored for ammonia, total organic carbon, chemical oxygen demand and suspended solids plus a range of trace metals, pesticides and PCBs. These non-GLP facility background data, carried out by ALS Life Sciences Limited, are shown in Appendix 3 (attached).

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: The photoperiod used was 16 hours of light: 8 hours of dark, with 20-minute dawn: dusk transition periods.
- Light intensity: Light intensity was measured at the start of the test at the aquaria water level in approximately the middle of the test rig and was determined to be 480 lux (cosine).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The number of live and dead embryos were recorded daily
Fry mortality, behaviour and appearance were made and recorded daily.
The survival, total length, and wet weight (blotted dry) were recorded along with the total dry weight of the population in each test vessel at the end of the exposure period.
Any physical abnormalities were also recorded at the end of the exposure period.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approx 2.2

RANGE-FINDING STUDY
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

no

Key result

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

0.455 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

33 d

Dose descriptor:

LOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

number hatched

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

length

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

weight

Remarks:

wet and dry

Details on results:

Behaviour and appearance
On Day 6 (1 day post-hatch), five fry (one in each of the nominal 0.043, 0.094 and 0.455 mg a.i./L concentrations and two in the nominal 1.0 mg a.i./L concentration) were found to be deformed. On Day 7 (2 days post-hatch), one fry in the dilution water control was found to have a bent spine. On Day 8 (3 days post-hatch), one fry in the nominal 0.043 mg a.i./L concentration was found to have a bent spine and one fry in the nominal 1.0 mg a.i./L concentration was found to have a bent spine and was swimming erratically upside down. Each fry was euthanised at the time the abnormality was observed for ethical reasons.
On Day 11 (6 days post-hatch), one fry in the dilution water control was recorded as significantly smaller than others in the tank but was behaving normally. Additionally, on the same day, one fry in the nominal 0.094 mg a.i./L concentration was found to have a bent spine, with lethargy and fungal growth and was euthanised at that time for ethical reasons. On Day 13 (8 days post-hatch), one fry in the nominal 0.043 mg a.i./L concentration was found to have a severely bent spine and was euthanised for ethical reasons.
On Day 22 (17 days post-hatch), one fry in the dilution water control was found to have a bent spine and was extremely small compared to the other fry and was seen swimming only on the bottom of the tank, not in the water column. The affected fry was euthanised for ethical reasons.
On Day 25 (20 days post-hatch), two fry in the dilution water control were recorded as much smaller in size compared to other fry but otherwise appeared healthy. On Day 26 (21 days post-hatch), one fry in the nominal 0.043 mg a.i./L concentration was found to have a bent spine and was swimming at the surface in a circular motion, the affected fry was euthanised for ethical reasons. On Day 33 (28 days post-hatch), one fry in the nominal 1.0 mg a.i./L concentration was found to be small, with a loss of orientation and was corkscrew swimming, the affected fry was euthanised as part of the test end procedures.
At the end of the test (28 days post-hatch), one fry from each of the nominal 0.094, 0.207 and 1.0 mg a.i./L concentrations were observed to have bent spines. These were not euthanised during the exposure as they otherwise appeared healthy (i.e. were of a similar size to the other fish, were feeding well and swimming normally). One fry in the nominal 1.0 mg a.i./L concentration was also observed to be blind with no observed pupil during the procedures at the end of the test.

Hatch data
All egg hatch data are shown in Table 3 below.
The range of percentage hatch success across all replicates in the test was 90 to 100%. The mean percentage hatch success across all replicates in the dilution water control was 98%. No significant differences (p <0.05) were found between the dilution water control and the treatment concentrations for hatch data (Chi2 2x2 Table Test with Bonferroni correction).
Probit analysis using linear maximum likelihood regression was conducted on the dilution water control and treatment hatch data, however the ECx values could not be determined due to the lack of a concentration response. In this case, the ECx values have been reported as >1.0 mg/L.
On Day 4 and Day 5, a number of fry were observed to have escaped from the egg cup into the test aquarium for 0.455 mg a.i./L replicate B. No action was taken as it was thought that, as hatch day was imminent, there was no meaningful difference regarding whether the hatched fry remained in the egg cup or had escaped into the aquarium.

Survival data
The survival data are shown in Table 3 below.
The range of percentage survival post-hatch across all replicates was 72 to 94%. The mean percentage survival post-hatch in the dilution water control was 90%. A significant difference (p > 0.05) was found between the dilution water control and the nominal 1.0 mg a.i./L concentration (Chi2 2x2 Table Test with Bonferroni correction).
The LC10 and LC20 values for survival (post-hatch) have not been reported as confidence limits could not be determined and the statistical report (Appendix 7 attached) indicates that, for the purposes of the point estimate determination, the slope of the relationship was not significantly different from zero and therefore the values were not considered to be reportable. For information only, the calculated values were LC10 = 0.171 mg a.i./L and LC20 = 0.883 mg a.i./L.
Probit analysis using linear maximum likelihood regression was conducted on the dilution water control and treatment survival data, however the LC10 and LC20 values could not be reported. The LC50 value could not be determined due to the lack of a concentration response at this level. In this case, the LC50 value has been reported as >1.0 mg/L.

Length data
The summary fry total length data are shown in Table 4 below.
The total lengths of the fry in the dilution water control ranged from 10.77 to 25.81 mm with a mean of 22.05 mm. The mean length of the dilution water control exceeded the 18 mm typical control minimum mean total length requirement of the OECD 210 TG. No significant differences (p <0.05) were found between the mean total length (per replicate) of the dilution water control and the nominal 0.043, 0.094, 0.207, 0.455 and 1.0 mg a.i./L test concentrations (Multiple Sequentially-rejective Welch t-test after Bonferroni-Holm correction).
Determination of ECx values was not required as the mean total length for each of the treatment concentrations was greater than the dilution water control, indicating that there were no adverse effects caused by the test substance.

Weight data
The summary fry wet weight data are shown in Table 5 below.
The wet weights of the fry in the dilution water control ranged from 0.00961 to 0.16421 g with a mean of 0.10388 g. No significant differences (p <0.05) were found between the wet weight in the dilution water control and the nominal 0.043, 0.094, 0.207, 0.455 and 1.0 mg a.i./L test concentrations (Multiple Sequentially-rejective Welch t-test after Bonferroni-Holm correction).
A non-linear regression was conducted on the dilution water control and treatment wet weight data however the ECx values could not be determined due to the lack of a concentration response. In this case, the ECx values have been reported as >1.0 mg/L.
The summary dry weight data are shown in Table 6 below. The calculated dry weights of individual fry (estimated using the dry weight of the pooled fry from each replicate) in the dilution water control ranged from 0.01863 to 0.02279 g with a mean of 0.02130 g. No significant differences (p <0.05) were found between the dry weight of the dilution water control and the nominal 0.043, .0.094, 0.207, 0.455 and 1.0 mg a.i./L test concentrations (Multiple Sequentially-rejective Welch t-test after Bonferroni-Holm correction).
Determination of ECx values was not required as the mean dry weight for each of the treatment concentrations was greater than the dilution water control, indicating that there were no adverse effects caused by the test substance.

Analytical data

The measured concentrations of the four major individual components of the test substance (C12, C14, C16 and C18-α-sulfo, 1-methyl esters, sodium salts) in the test solutions and the fortified solutions are given in Table 1 and Table 2, respectively (attached). Typical chromatograms are shown in Appendix 5 (attached), Figures 1-10. Typical calibration curves are shown in Appendix 5, Figure 11. All analytical values are quoted to three significant figures and percentages to the nearest integer. The measurement of the C16-α-sulfo, 1-methyl esters, sodium salts component using the analytical method (Appendix 5) did not meet the acceptance criteria of the method validation study.

A single replicate test vessel from each treatment was sampled and measured at each time point. Samples of the dosing stock solutions were taken and measured on Day -3 (pre-exposure) and the results are given in Table 1 (attached). The limit of quantification of Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts was 10 μg/L taken from the method validation study.

The dilution water control was measured and determined to be <LOQ for all measured components throughout the exposure. Measured concentrations of the four major individual components during the exposure (Table 1) were as follows:

- C12-α-sulfo, 1-methyl esters, sodium salts ranged between 60-98% of nominal;

- C14-α-sulfo, 1-methyl esters, sodium salts ranged between 57-102% of nominal;

- C16-α-sulfo, 1-methyl esters, sodium salts ranged between 55-132% of nominal;

- C18-α-sulfo, 1-methyl esters, sodium salts ranged between 55-314% of nominal.

The variability of the measured concentrations in the test aquaria was thought to be due to the properties of the UVCB test substance as the dosing stock solution and dilution water flow rates into the mixing chambers were confirmed to be within ±10% at each timepoint throughout the test (with exceptions as noted). Additionally, the concentrations of the four major and four minor components of the test substance were measured in the dosing stock solutions on Day -3 (pre-exposure) and ranged between 86-120% of nominal (Table 1 and Appendix 6(attached), respectively).

Measured concentrations of the fortified recovery samples for the four major individual components during the exposure (Table 2) were as follows:

- C12-α-sulfo, 1-methyl esters, sodium salts ranged between 81-111% of nominal;

- C14-α-sulfo, 1-methyl esters, sodium salts ranged between 87-110% of nominal;

- C16-α-sulfo, 1-methyl esters, sodium salts ranged between 87-112% of nominal;

- C18-α-sulfo, 1-methyl esters, sodium salts ranged between 89-117% of nominal.

As multiple components of the test substance were measured, nominal concentrations were used for calculation and reporting of results, with measured concentrations for each individual component presented separately.

Measured concentrations of the four minor individual components of the test substance (C12, C14, C16 and C18-α-sulfo, disodium salts) in the test solutions and the fortified recovery samples are given in Appendix 6.

Table 3 Hatch and survival data

Nominal concentration of Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts (mg a.i./L)	Replicate	Eggs introduced on Day 0	Total number hatched	% Hatched		Number of fry surviving	% Survival post-hatch
				Replicate	Treatment		Replicate	Treatment
Dilution water control	A	20	18	90	98	17	94	90
	B	20	20	100		18	90
	C	20	20	100		19	95
	D	20	20	100		16	80
0.043	A	20	20	100	98	20	100	91
	B	20	20	100		20	100
	C	20	19	95		15	79
	D	20	19	95		16	84
0.094	A	20	20	100	100	16	80	94
	B	20	20	100		20	100
	C	20	20	100		19	95
	D	20	20	100		20	100
0.207	A	20	20	100	99	18	90	94
	B	20	19	95		16	84
	C	20	20	100		20	100
	D	20	20	100		20	100
0.455	A	20	20	100	98	17	85	90
	B	20	19	95		18	95
	C	20	19	95		18	95
	D	20	20	100		17	85
1.0*	A	20	20	100	99	15	75	72
	B	20	20	100		12	60
	C	20	20	100		13	65
	D	20	19	95		17	89

No statistically significant (p <0.05) differences between the dilution water control and the treatment groups were observed for the Hatch endpoint.

*A statistically significant (p >0.05) difference between the dilution water control and the nominal 1.0 mg a.i./L concentration was observed for the Survival (post-hatch) endpoint.

Percentages are quoted to the nearest integer.

Table 4 Summary total length data

Nominal concentration of Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts (mg a.i./L)	Replicate	Number of fry	Mean total length (mm)	Minimum total length (mm)	Maximum total length (mm)	Standard deviation (mm)
Dilution water control	A	17	21.62	18.79	25.16	1.7
	B	18	22.17	20.00	25.70	1.4
	C	19	22.63	18.13	25.58	2.0
	D	16	21.71	10.77	25.81	4.5
	Pooled	70	22.05	10.77	25.82	2.6
0.043	A	20	22.47	18.64	24.97	1.3
	B	20	22.52	17.86	25.23	1.8
	C	15	22.32	14.84	25.15	2.5
	D	16	22.51	13.41	25.17	2.8
	Pooled	71	22.46	13.41	25.23	2.1
0.094	A	16	22.73	18.33	25.84	1.9
	B	20	22.88	13.40	26.76	3.6
	C	19	22.58	20.12	25.84	1.8
	D	20	22.28	14.97	25.76	2.6
	Pooled	75	22.61	13.40	26.76	2.6
0.207	A	18	22.81	18.72	25.74	1.8
	B	16	22.95	18.24	25.73	1.9
	C	20	23.51	21.36	26.10	1.4
	D	20	22.97	20.19	24.93	1.4
	Pooled	74	23.07	18.24	26.10	1.6
0.455	A	17	23.44	20.66	26.28	1.6
	B	18	22.88	16.76	26.14	2.6
	C	18	23.44	18.29	27.28	2.2
	D	17	23.83	20.00	26.48	1.8
	Pooled	70	23.39	16.76	27.28	2.1
1.0	A	15	22.85	20.55	24.44	1.1
	B	12	21.98	15.35	25.19	2.9
	C	13	23.84	18.48	27.22	2.6
	D	17	24.12	21.61	26.16	1.3
	Pooled	57	23.27	15.35	27.22	2.1

No statistically significant (p <0.05) differences between the dilution water control and the treatment groups were observed for the total length endpoint.

Lengths reported to 2 decimal places. Pooled means calculated using individual fry length values.

Table 5 Summary wet weight data

Nominal concentration of Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts (mg a.i./L)	Replicate	Number of fry	Mean wet weight (g)	Minimum wet weight (g)	Maximum wet weight (g)	Standard deviation (g)
Dilution water control	A	17	0.09460	0.05289	0.15084	0.02712
	B	18	0.10736	0.07451	0.16421	0.02189
	C	19	0.10832	0.04852	0.14960	0.02973
	D	16	0.10453	0.00961	0.15274	0.04314
	Pooled	70	0.10388	0.00961	0.16421	0.03088
0.043	A	20	0.10879	0.05187	0.14528	0.02114
	B	20	0.10499	0.04922	0.14638	0.02409
	C	16	0.10616	0.02738	0.14798	0.02848
	D	15	0.10452	0.01929	0.15303	0.03199
	Pooled	71	0.10623	0.01929	0.15303	0.02569
0.094	A	16	0.11360	0.05735	0.15998	0.02942
	B	20	0.11295	0.02033	0.17750	0.03924
	C	19	0.10469	0.06501	0.15262	0.02562
	D	20	0.09633	0.02161	0.14691	0.03305
	Pooled	75	0.10656	0.02033	0.17750	0.03259
0.207	A	18	0.10256	0.04623	0.15050	0.02701
	B	16	0.10535	0.04708	0.14497	0.02476
	C	20	0.10904	0.08044	0.15593	0.01973
	D	20	0.09820	0.05720	0.13055	0.01895
	Pooled	74	0.10374	0.04623	0.15593	0.02253
0.455	A	17	0.10765	0.06821	0.14682	0.02172
	B	18	0.09933	0.03661	0.14934	0.03355
	C	18	0.10955	0.04801	0.16251	0.02988
	D	17	0.11145	0.05785	0.15155	0.02758
	Pooled	70	0.10692	0.03661	0.16251	0.02837
1.0	A	15	0.09776	0.05640	0.12957	0.01886
	B	12	0.08450	0.01883	0.12676	0.03475
	C	13	0.12298	0.05266	0.15910	0.03323
	D	17	0.11519	0.08482	0.14671	0.01774
	Pooled	57	0.10592	0.01883	0.15910	0.02937

No statistically significant (p <0.05) differences between the dilution water control and the treatment groups were observed for the wet weight endpoint.

Wet weights reported to 5 decimal places. Pooled means calculated using individual fry wet weight values.

Table 6 Dry weight data

Nominal concentration of Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts (mg a.i./L)	Replicate	Mean dry weight per fry (g)	Treatment mean dry weight per fry (g)	Standard deviation (g)
Dilution water control	A	0.01863	0.02130	0.00183
	B	0.02186
	C	0.02279
	D	0.02193
0.043	A	0.02251	0.02234	0.00119
	B	0.02166
	C	0.02124
	D	0.02394
0.094	A	0.02392	0.02430	0.00185
	B	0.02471
	C	0.02205
	D	0.02651
0.207	A	0.02201	0.02234	0.00138
	B	0.02216
	C	0.02423
	D	0.02094
0.455	A	0.02415	0.02383	0.00111
	B	0.02218
	C	0.02458
	D	0.02441
1.0	A	0.02040	0.02339	0.00403
	B	0.01949
	C	0.02749
	D	0.02617

No statistically significant (p <0.05) differences between the dilution water control and the treatment groups were observed for the dry weight endpoint.

Validity criteria

The study validity criteria were as follows:

1. The dissolved oxygen concentration must be at least 60% of the air saturation value throughout the test.

In this study, the minimum observed DO concentration was 75.2% (Table 8; attached).

 

2. Constant conditions should be maintained as far as possible throughout the test and the water temperature should not differ by more than ±1.5ºC between test vessels or between successive days at any time during the test and should be within the temperature range specified for the test species of 25 ± 1.5ºC.

The temperature of the test solutions ranged from 23.4 to 25.2°C. The maximum difference between test vessels at a single time point was 1.5°C on Day 5 (hatch day) (Table 9; attached). The continuous temperature measurement, recorded in DWC Replicate D ranged from 24.4 to 25.9°C (Table 10; attached).

 

3. Concentrations of Fatty acids, C12-18(even numbered)-methyl esters, sulfonated, sodium salts must be determined. When concentrations remain within 80-120% of nominal, the effect concentrations can be expressed relative to nominal or measured concentrations. If this is not the case, the results should be based on measured concentrations.

In this study, concentrations of eight individual components of the test substance were measured (three major components which the analytical method was validated for and one major and four minor components for which the analytical method was not validated). The concentrations of these components did not remain within 80-120% of nominal, however there was no clear method of generating a single mean measured concentration for each treatment. Therefore, nominal concentrations were used for the calculation and reporting of results with measured major and minor component concentrations presented in Table 1 (attached) and Appendix 6 (attached), respectively. This approach did not meet the criterion as described, however it was deemed to be the most suitable for this UVCB test substance.

 

4. Overall survival of fertilised eggs and post-hatch success in the control should be ≥70% and ≥75% respectively.

In this study the hatching success in the dilution water control was 98%. The post-hatch survival in the dilution water control was 90% (Table 3; attached).

Validity criteria fulfilled:

yes

Remarks:

(see any other information on results)

Conclusions:

Including all of the assessed endpoints at the end of the study, and using the nominal concentrations, the overall LOEC for Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts in this study was 1.0 mg a.i./L, and the overall NOEC was 0.455 mg a.i./L.

Executive summary:

Subject: Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts: Determination of effects on the Early Life-Stage of the fathead minnow (Pimephales promelas)

Guideline: OECD Test Guideline (TG; 2013), Test No. 210: Fish, Early-life Stage Toxicity Test

Test species: Newly fertilised (<24 hours old) eggs of Pimephales promelas

Source of organisms: Eggs supplied from broodstock held at Scymaris Ltd

Test concentrations: Dilution water control (DWC) and nominal concentrations of 0.043, 0.094, 0.207, 0.455 and 1.0 mg a.i./L

Test design: Flow-through dosing design with 4 replicates per treatment. Exposure duration: egg to 28 days post-hatch

Nominal test temperature: 25±1.5°C

Results based on nominal concentrations of Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts:

Survival (post-hatch): NOEC = 0.455 mg a.i./L; LOEC = 1 mg a.i./L

Growth (length): NOEC ≥ 1 mg a.i./L; LOEC > 1 mg a.i./L

Growth (wet weight): NOEC ≥ 1 mg a.i./L; LOEC > 1 mg a.i./L

Growth (dry weight): NOEC ≥ 1 mg a.i./L; LOEC > 1 mg a.i./L

Description of key information

33-d NOEC = 0.455 mg a.i./L (nominal) based on post-hatch survival (Pimephales promelas; OECD TG 210)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

0.455 mg/L

Additional information

The chronic toxicity of the registered substance to the early-life stages of the fathead minnow (Pimephales promelas) was determined in a GLP study performed according to OECD TG 210.

Newly fertilised (<24 hours old) eggs of Pimephales promelas were exposed to the registered substance to 28 days post hatch (33 days in total) at nominal concentrations of 0 (dilution water control) 0.043, 0.094, 0.207, 0.455 and 1.0 mg a.i./L. A flow-through dosing design was used with 4 replicates per treatment. The test was performed at a nominal temperature of 25 ± 1.5°C. 

To confirm the actual exposure concentrations being achieved, the concentrations of eight components of the registered substance in the test solutions were measured using a LC-MS/MS method. Measurement of three of the major individual components of the registered substance (C12, C14 and C18-α-sulfo, 1-methyl esters, sodium salts) using this method was validated in a separate study. The method also measured C16-α-sulfo, 1-methyl esters, sodium salts (which did not meet the acceptance criteria of the method validation study) and four of the minor individual components of the registered substance (C12, C14, C16 and C18-α-sulfo, disodium salts) but measurement of these components using this method was not validated. 

A single replicate test vessel from each treatment was sampled and measured at each time point. Samples of the dosing stock solutions were taken and measured on Day -3 (pre-exposure). 

Measured concentrations of the four major individual components during the exposure were as follows:

- C12-α-sulfo, 1-methyl esters, sodium salts ranged between 60-98% of nominal;

- C14-α-sulfo, 1-methyl esters, sodium salts ranged between 57-102% of nominal;

- C16-α-sulfo, 1-methyl esters, sodium salts ranged between 55-132% of nominal;

- C18-α-sulfo, 1-methyl esters, sodium salts ranged between 55-314% of nominal. 

The variability of the measured concentrations in the test aquaria was thought to be due to the properties of the UVCB test substance as the dosing stock solution and dilution water flow rates into the mixing chambers were confirmed to be within ±10% at each timepoint throughout the test (with exceptions as noted). Additionally, the concentrations of the four major and four minor components of the test substance were measured in the dosing stock solutions on Day -3 (pre-exposure) and ranged between 86-120% of nominal. 

Measured concentrations of the fortified recovery samples for the four major individual components during the exposure (Table 2) were as follows:

- C12-α-sulfo, 1-methyl esters, sodium salts ranged between 81-111% of nominal;

- C14-α-sulfo, 1-methyl esters, sodium salts ranged between 87-110% of nominal;

- C16-α-sulfo, 1-methyl esters, sodium salts ranged between 87-112% of nominal;

- C18-α-sulfo, 1-methyl esters, sodium salts ranged between 89-117% of nominal. 

As multiple components of the test substance were measured, nominal concentrations were used for calculation and reporting of results, with measured concentrations for each individual component presented separately. 

The range of percentage hatch success across all replicates in the test was 90 to 100%. The mean percentage hatch success across all replicates in the dilution water control was 98%. No significant differences (p <0.05) were found between the dilution water control and the treatment concentrations for hatch data (Chi2 2x2 Table Test with Bonferroni correction).

The range of percentage survival post-hatch across all replicates was 72 to 94%. The mean percentage survival post-hatch in the dilution water control was 90%. A significant difference (p > 0.05) was found between the dilution water control and the nominal 1.0 mg a.i./L concentration (Chi2 2x2 Table Test with Bonferroni correction). 

The total lengths of the fry in the dilution water control ranged from 10.77 to 25.81 mm with a mean of 22.05 mm. The mean length of the dilution water control exceeded the 18 mm typical control minimum mean total length requirement of the OECD 210 TG. No significant differences (p <0.05) were found between the mean total length (per replicate) of the dilution water control and the nominal 0.043, 0.094, 0.207, 0.455 and 1.0 mg a.i./L test concentrations (Multiple Sequentially-rejective Welch t-test after Bonferroni-Holm correction).

The wet weights of the fry in the dilution water control ranged from 0.00961 to 0.16421 g with a mean of 0.10388 g. No significant differences (p <0.05) were found between the wet weight in the dilution water control and the nominal 0.043, 0.094, 0.207, 0.455 and 1.0 mg a.i./L test concentrations (Multiple Sequentially-rejective Welch t-test after Bonferroni-Holm correction). 

The calculated dry weights of individual fry (estimated using the dry weight of the pooled fry from each replicate) in the dilution water control ranged from 0.01863 to 0.02279 g with a mean of 0.02130 g. No significant differences (p <0.05) were found between the dry weight of the dilution water control and the nominal 0.043, .0.094, 0.207, 0.455 and 1.0 mg a.i./L test concentrations (Multiple Sequentially-rejective Welch t-test after Bonferroni-Holm correction).

Based on these findings, the following NOEC/LOEC were reported:

Survival (post-hatch): NOEC = 0.455 mg a.i./L; LOEC = 1 mg a.i./L

Growth (length): NOEC ≥ 1 mg a.i./L; LOEC > 1 mg a.i./L

Growth (wet weight): NOEC ≥ 1 mg a.i./L; LOEC > 1 mg a.i./L

Growth (dry weight): NOEC ≥ 1 mg a.i./L; LOEC > 1 mg a.i./L