Alcohols, C8-10, ethoxylated, sulfates, ammonium 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:

10 October - 05 December 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)

Version / remarks:

adopted July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples (5 mL) were taken from the control and test concentration prior to organism addition, on Day 0 (organism addition) and on Days 3, 11, 18, 25 and 28 post-hatch for analysis.
- Sampling method: The samples were taken using an air displacement pipette fitted with a plastic pipette tip into 20 mL glass scintillation vials containing 5 mL of methanol

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: ca 4092 mg of test substance was dissolved in 11 L of treated mains water to give a 125 mg a.i./mL stock solution. The stock solution was prepared and replaced daily throughout the duration of the test.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Source: eggs used in this study were obtained from an in-house laboratory breeding system

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): Viable fish eggs were obtained from breeding groups of Pimephales promelas. Prior to initiation of the test, the stage of development was established.
- Method of collection of fertilised eggs: < 24 hours prior to testing, collected in glass scintillation vials containing dilution water

POST-HATCH FEEDING
- Type/source of feed: brine shrimp (Artemia salinis) nauplii, freshly cultured during the test
- Amount given: initially the larvae were fed approximately 24-hour old brine shrimp. From Day 7 post-hatch onwards the larvae were fed approximately 48-hour old brine shrimp.

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

28 d

Test temperature:

24.1 - 25.1°C

pH:

6.85 - 7.86

Dissolved oxygen:

88.4 - 103.6% ASV

Nominal and measured concentrations:

Nominal test substance concentration: 25 mg a.i./L
Mean measured test concentration: 11.2 mg/L

Details on test conditions:

TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): 17 cm long glass cylinders (6.5 cm internal diameter) with a 0.5 - 1.0 mm non-reactive mesh attached to one end (bottom).
- Test vessel: 3 L glass aquaria
- Fill volume: 2.5 L
- Type of flow-through (e.g. peristaltic or proportional diluter): the stock solution was dosed at a rate of 7 ± 10% mL/minute to a diluent flow rate of 28 ± 10% mL/minute through an in-line mixer
- Renewal rate of test solution (frequency/flow rate): five total replacements within 24 h
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was treated mains water (municipal water treated via activated carbon particulate filter and ultraviolet irradiation).
- Intervals of water quality measurement: weekly measurement of pH and dissolved oxygen, daily measurement of temperature

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light:8 hour dark period with an approximate 30 minute dawn:dusk transition period.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : On Day 28 post-hatch the total numbers of surviving larvae were counted and individual total fish length and wet weight of all remaining fish were determined.

RANGE-FINDING STUDY
- Test concentrations: 0.25, 2.5, 25 mg a.i./L
- Results used to determine the conditions for the definitive study: 100% post hatch survival

POST-HATCH DETAILS
- Begin of post-hatch period: day 6

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

11.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: time-to-hatch, hatching success, normal larvae at hatch, post-hatch survival and growth

Remarks on result:

other: Concentration based on eight representative constituents

Details on results:

- Post-hatch survival: 91% in controls and treatment, respectively
- Days to hatch or time to release of young: First egg hatch occurred between Day 3s and 4 and completion of hatching occurred between Days 4 and 6 (post-egg addition) in the test concentration and the control vessels.
- Numbers hatched: control 96%, treatment 90%
- Observations on body length and weight of young and/or exposed parents at one or more time periods: The mean fish total length in the control and test concentration groups was 19.5 and 19.6 mm, respectively.
The mean fish wet weight in the control and test concentration groups was 67.3 and 62.0 mg, respectively.
- Type of and number with morphological abnormalities: 3 larvae of the control with bent spine and 1 larvae of the test concentration was observed to be underdeveloped. These observations were not concentration specific.
- Type of and number with behavioural abnormalities: none
- Type and number of developmental effects: none
- Type and magnitude of hormonal changes: none

Reported statistics and error estimates:

Statistical analysis of the data was performed using the CETIS program v 1.8.6.8.
The Lowest Observed Effect Concentration (LOEC) is defined as the lowest tested concentration at which the chemical is observed to have a statistically significant effect compared to the control. The No Observed Effect Concentration (NOEC) is the test concentration immediately below the LOEC.
To confirm no significant effects were observed between the control and test concentration, statistical analysis was conducted for the hatching success, post-hatch survival and growth (wet weight and length) endpoints.
Each NOEC and LOEC analysis included Bartletts Equality of Variance test and Shapiro-Wilks Test to evaluate homogeneity and normality of the data, respectively. In addition, Grubbs Extreme Value test was also included to determine the presence of outliers. The data were not transformed prior to analysis.
To determine the NOEC and LOEC values, the number of hatched larvae and the number of larvae surviving at 28 days post-hatch data for the test concentration were compared to the control using the Fisher Exact Test. The Day 28 length and wet weight data for the test concentration were compared to the control using the Equal Variance t Two-Sample Test. All data were shown to be homogenous and normally distributed. No statistical outliers were detected in the data sets.
Given that no statistically significant effects were observed between the control and test substance group and that the test was conducted using a single test concentration, the ECx values could not be calculated.

Table 1: Biological results

Measured concentration [mg/L]	Vessel number	Number of eggs added at start of test	Number of hatched larvae	Number of normal larvae at hatch	Number of larvae surviving at 28 days post-hatch	Mean Total length of fish [mm] at 28 days post-hatch	Mean Wet weight of fish [mg] at 28 days post-hatch
Control	V1	20	20	18	15	20.4	76.1
	V2	20	19	19	19	19.0	60.6
	V3	20	19	19	18	19.6	69.4
	V4	20	19	19	18	19.0.	63.2
11.2	V1	20	20	20	20	18.9	53.7
	V2	20	16	16	13	20.6	73.1
	V3	20	19	19	17	20.0	56.7
	V4	20	17	17	16	19.0	64.6

Table 2: Analysis of test media per constituent during the definitive test

Constituent analysed	Measured concentration [mg/L]
	Day 0		Day 11 (post-hatch)		Day 18 (post-hatch)	Day 25 (post-hatsch)	Day 28 (post-hatch)
Ammonium octyl sulfate	0.612	0.739		0.462		0.496		0.181
1-Octanol, ethoxylated (1 EO), sulfated, ammonium salt	1.35	1.14		0.796		0.766		0.459
1-Octanol, ethoxylated (2 EO), sulfated, ammonium salt	1.52	1.42		1.39		1.51		1.10
1-Octanol, ethoxylated (3 EO), sulfated, ammonium salt	1.41	1.37		1.21		1.29		1.02
Ammonium decyl sulfate	0.286	0.235		0.233		0.304		0.0750
1-Decanol, ethoxylated (1 EO), sulfated, ammonium salt	0.439	0.426		0.352		0.448		0.244
1-Decanol, ethoxylated (2 EO), sulfated, ammonium salt	1.31	1.27		1.02		0.890		0.626
1-Decanol, ethoxylated (3 EO), sulfated, ammonium salt	1.21	1.16		0.821		0.663		0.486
Sum of constituents	8.14	7.76		6.28		6.37		4.19
Test substance concentration^a	13.9	13.3		10.7		10.9		7.16

a= Calculated based on sum of the measured concentrations of each constituent x 1.71 (correction factor based on measured constituents representing 58.4% of total test substance)

Table 2: 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.	88.4 - 103.6%	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).	≤1°C	yes
The analytical measure of the test concentrations is compulsory.	Analytical monitoring by LC-MS/MS	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.	The mean hatching success of embryos in the control group was 96% which was in excess of the validity criterion of ≥70%. The mean post-hatch survival in the control group was 91% which was greater than the validity criterion of ≥75%.	yes

Validity criteria fulfilled:

yes

Remarks:

Please refer to table 3 at "Any other infrmation on results incl. tables"

Description of key information

NOEC (28 d): 11.2 mg/L (mean measured, OECD 210, Pimephales promelas)

Additional information

The assessment is based on the data currently available. New studies, based on the category review and the final decisions issued for some of the category substances, which are also relevant for this assessment, are currently being conducted. The hazard assessment with respect to aquatic toxicity will be updated once all ongoing studies have been finalised.

The chronic toxicity of the substance to fish was tested in a GLP guideline study conducted according to OECD 210. The test was conducted as a limit test under flow-through conditions. Fertilised eggs of the freshwater fish species Pimephales promelas were exposed to a nominal limit test item concentration of 25 mg a.i./L. The substance concentrations of the UVCB substance was analytically verified by targeting eight different constituents in the LC-MS/MS analysis. The overall mean measured concentration was 11.2 mg/L. No effects on hatching success, post-hatch survival, final fish length and final fish wet weight were observed. The determined NOEC was 11.2 mg/L.