1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-C12-14 acyl derivs., hydroxides, inner salts

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 June to 27 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

Deviations:

yes

Remarks:

Additional samples were taken on Days 4 and 5 from 0.32 mg solid content/L after a failure in the dosing system. Results of these additional measurements were considered with the mean exposure concentration calculation and a worst-case scenario followed.

GLP compliance:

yes

Specific details on test material used for the study:

Appearance: Yellow liquid
Purity/Composition: UVCB – 49.7% (solids)
Test item storage: In refrigerator (2-8°C)
Stable under storage conditions until: 15 March 2019 (expiry date)

Analytical monitoring:

yes

Details on sampling:

Samples (1.5 mL) were taken from one replicate of each test group and the control, changing systematically amongst replicates, for possible chemical analysis. Samples were taken one day before the start of exposure to check the functioning of the system. Samples were then taken at the start of testing, and after 7, 14, 21, 28 and 32 days of exposure. Samples were also taken on Day 4 and Day 5 from 0.32 mg solid content/L after a failure in the dosing system. Samples that were not analysed on the day of sampling were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility. Additionally, reserve samples of 1.5 mL were taken from all test solutions, the stock solutions and on Day 28 also from the mixing flasks for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

The test item was a liquid UVCB with a solid content of 49.7%. A correction was made for the solid content of the UVCB. All further concentrations reported are based on solid content.

Fresh test item stock solutions of 100 mg solid content/L (i.e. 200 mg of the UVCB/L) were prepared in Milli-RO water every 48 hours, and exact volumes of these test item stock solutions were dosed with syringes via a computer-controlled system consisting of six dispensers (Gilson). All stock solutions were clear and colourless. The dosed volumes of the stock solution entered a mixing flask separately from the dilution water supply (adjusted ISO-medium). The dilution water was supplied applying flow-meters at a constant rate of 5 litres per hour.

In the mixing flask the dosed volume and the dilution water were mixed under continuous stirring. Peristaltic pumps, set at a rate of 1 litre per hour, were used to divide the contents of the mixing vessels continuously and equally over four replicate test vessels containing the embryos/larvae/juvenile fish. The flow meters were calibrated before the start of the exposure and weekly thereafter. The whole system was checked twice daily. See 'Any other information on materials and methods incl. tables' for details on the dosing system used.

Test organisms (species):

Pimephales promelas

Details on test organisms:

Test organisms source: In house culture

Brood stock:
Brood stock were held in adjusted ISO medium, formulated using RO-water with the following composition:
- CaCl2.2H2O 211.5 mg/L
- MgSO4.7H2O 88.8 mg/L
- NaHCO3 46.7 mg/L
- KCl 4.2 mg/L
Measurements of conductivity, pH, nitrate, nitrite and ammonia concentration were taken once a week, and temperature measured continuously. Temperature was also measured before transferring the parental fish to the breeding system. Water quality parameters were kept within the optimum limits for the respective fish species. The male/female ratio was 1:2. Fish were provided a diet of frozen brine shrimp Nauplii and pelleted fish food (SDS 400, Coppens International bv, Helmond, The Netherlands). At the time of fertilisation, males and females were put together in spawning tanks, with spawning starting the following day approximately 1 to 2 hours after lights have been switched on. The spawning tank was equipped with a substrate (pvc-tube), enabling collection of fertilised eggs.

Test organisms:
Embryos were introduced into test systems (stainless steel test vessels) before cleavage of the blastodisc commenced (approximately 2-4 hours after fertilisation). No feeding occurred during the embryonic phase. Larvae and juvenile fish were fed ad libitum with brine shrimp Nauplii 24 or 48-hours old.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

32 d

Post exposure observation period:

Not applicable

Hardness:

161 to 196 mg CaCO3/L

Test temperature:

Average temperature measured at weekly intervals in the control and the treatment groups: 24.4 to 25.2°C
Temperature continuously measured in one of the control replicates: 24.7 to 25.7°C

pH:

7.4 to 8.1

Dissolved oxygen:

6.3 to 8.6 mg O2/L

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 0.010, 0.032, 0.10, 0.32 and 1.0 mg solid content/L
Mean measured concentrations: 0 (control), 0.0082, 0.020, 0.072, 0.19 and 1.0 mg solid content/L

Test concentrations were selected based on the results of a range finding test.

Details on test conditions:

Tests were conducted in stainless steel vessels (~1.7L) with an approximate exposure volume of 1.5L; covered with a Perspex plate (open test system), and the test medium was adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3. The experiment started on nominal Day 0 with 80 fresh and healthy fathead minnow embryos per test group. Embryos were randomly distributed and divided equally over four stainless steel vessels. Tests were conducted under a 16 h photoperiod daily, between 726 and 805 lux. Test solutions were not aerated.


The following measurements and recordings were made throughout the study:

Test media
Oxygen, pH and temperature: At the start of the test, at weekly intervals and then at the end of the test in all replicates of each test group. Temperature was also recorded continuously in one of the control replicates.
Hardness: At the start and the end of the test in one replicate of each test group.
Total Organic Carbon (TOC): At the start of the test in blank-medium.

Test organisms
Stage of embryonic development: At the beginning of exposure from at least a representative sample of eggs.
Hatching and survival: Numbers of hatched larvae and dead embryos, larvae and juvenile fish were recorded daily (dead embryos, larvae and juvenile fish were removed directly after recording).

Criteria for death
Eggs: A marked loss of translucency and change in coloration, caused by coagulation and/or precipitating of protein, characterised by a white opaque appearance.
Embryos: Absence of body movement and/or absence of heartbeat;
Larvae and juvenile fish: Immobility and/or absence of respiratory movement and/or absence of heartbeat and/or white opaque coloration of the central nervous system and/or lack of reaction to mechanical stimulus.

Abnormal appearance/behaviour: Abnormalities (e.g. hyperventilating, uncoordinated swimming, atypical quiescence and atypical swimming behaviour) were recorded daily.
Body weight: All surviving fish were weighed on a replicate basis (blotted dry weight) at the end of the test.
Body length: Individual lengths from the surviving fish were measured at the end of the test.


At the end of the test the surviving larvae were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

Reference substance (positive control):

not specified

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.072 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

number hatched

Duration:

32 d

Dose descriptor:

LOEC

Effect conc.:

0.019 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

number hatched

Duration:

32 d

Dose descriptor:

EC10

Effect conc.:

1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

number hatched

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.072 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

other: Post-hatch survival

Duration:

32 d

Dose descriptor:

LOEC

Effect conc.:

0.019 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

other: Post-hatch survival

Duration:

32 d

Dose descriptor:

EC10

Effect conc.:

0.097 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

other: Post-hatch survival

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.008 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

length

Duration:

32 d

Dose descriptor:

LOEC

Effect conc.:

0.02 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

length

Duration:

32 d

Dose descriptor:

EC10

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

length

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

< 0.008 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

weight

Duration:

32 d

Dose descriptor:

LOEC

Effect conc.:

0.008 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

weight

Key result

Duration:

32 d

Dose descriptor:

EC10

Effect conc.:

0.075 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

weight

Details on results:

Measured Concentrations:
Analyses of samples taken from the nominal concentrations on a weekly basis showed that measured concentrations were generally at or below nominal (44-116%). Measurement of concentrations in the mixing flasks on Day 28 and in the stock on Day-1 showed that the concentrations were in agreement with nominal, i.e. 98-110% in the mixing flasks and 109-116% in the stock. It was therefore expected that the lower concentrations in the test vessels was related to the presence of fish. Following a worst-case scenario, the effect parameters were based on mean measured concentrations.

Embryonic survival and hatching:
The overall survival of embryos at the end of hatching was 98% for the control, which complies with the requirements of the guideline (i.e. at least 70% of the embryos in the control should hatch). Hatching success was 98-100% in concentrations up to and including 0.072 mg solid content/L and not statistically different from the control. Hatching success at the two highest concentrations of 0.19 and 1.0 mg solid content/L was 93 and 90%, respectively, but proved to be statistically different from the control. The test item caused earlier onset of hatching. First larvae were observed on day three of exposure and were mainly related to the two highest concentrations tested (i.e. 25 and 31%, of the introduced embryos, respectively, had hatched). On day four the percentage of hatching in all five test concentrations was 9, 29, 49, 71 and 86%, while hatching in the control did not start until day 5. Hatching was complete for the control and all test concentrations on day five of exposure.

Larval survival and development:
The mean post-hatch larval survival was 96% in the control. The validity criterion for posthatch survival of at least 75% was met. The survival rates ranged from 0 to ≥96% and were clearly dose-related. Statistical analyses indicated that larval survival was not statistically different from the control at test concentrations up to and including 0.072 mg solid content/L. Statistical analysis was not performed on abnormalities but based on the observations made during the test it appears that the severity and incidence of abnormalities were concentration dependent.

Effects on Larval Growth:
A statistically significant reduction of length was observed at concentrations of 0.020 mg solid content/L and higher, while reduction of weight was statistically significant at all concentrations tested.

All of the prescribed guideline validity criteria were met. Dissolved oxygen concentration was maintained above 60% of the air saturation value throughout the test. Water temperature did not differ by more than ± 1.5°C between test chambers between successive days at any time during the test, and was within the temperature ranges specified for the test species. Exposure concentrations during the test period were analytically monitored and effect parameters were based on mean measured concentrations. Overall survival of fertilised eggs in the control was >70% until hatching was complete (i.e. 98%). The overall survival of hatched larvae was >75% in the control during the remaining test period (i.e. 96%).

Results with reference substance (positive control):

Not reported

Reported statistics and error estimates:

The following statistical procedures were used to determine the NOEC and LOEC for hatching success, post-hatch survival, body weight and body length.

Hatching success and Post-hatch survival: Step-down Cochran-Armitage Test Procedure (α=0.05, one-sided greater).
Body weight and body length:
- Data distribution: Shapiro-Wilk´s Test
- Homogeneity of variance: Levene´s Test (with Residuals)
- Differences between treatments and the pooled control: Williams Multiple Sequential t-test Procedure (α=0.05, one-sided smaller).

Calculation of ECx-values was based on probit analysis using linear maximum likelihood regression with the percentages of post-hatch mortality and the percentages of reduction of growth (both weight and length) versus the logarithms of the corresponding average exposure concentrations of the test item.

All analyses were performed with ToxRat Professional 3.2.1 (ToxRat Solutions® GmbH, Germany).

Nominal, measured and mean measured concentrations during the ELS test

Nominal concentration Test item (mg solid content/L)	Measured concentration (mg solid content/L)								Mean conc. Test item (mg solid content/L)
	Day 0	Day^14	Day^15	Day 7	Day 14	Day 21	Day 28	Day 32
0.010	0.0116	n.m.	n.m.	0.0063	0.00898	0.0074	0.0068	0.0079	0.0082
0.032	0.0337	n.m.	n.m.	0.0181	0.0190	0.0151	0.0141	0.0192	0.020
0.10	0.108	n.m.	n.m.	0.0668	0.0765	0.0685	0.0578	0.0526	0.072
0.32	0.336	0.00873	0.000047	0.254	0.278	0.257	0.220	0.205	0.19
1.0	1.09	n.m.	n.m.	0.920	n.m.	n.m.	n.m.	n.m.	1.0

n.m. Not measured

¹Samples taken after a dosing failure that lasted for 16 hours on Day 4 and for at least 1¼ hours on Day 5

 

 

Post-Hatch Survival at the End of Exposure (Day 32)

Conc. (mg solid content/L)	Total Hatched	Survived	Dead	% Post-hatch Mortality
Control	78	75	3	3.8
0.0082	78	76	2	2.6
0.020	80	77	3	3.8
0.072	78	75	3	3.8
0.19	74	40	34	45.9*
1.0	72	0	72	100.0*

* Effect was statistically significant (p≤0.05)

 

 

Mean body length (mm) and percentage of reduction at the end of exposure

Conc. (mg solid content/L)	Mean	Std. Dev.	n	% Reduction
Control	25.38	0.386	4
0.0082	25.08	0.369	4	1.2
0.020	24.80	0.258	4	2.3*
0.072	24.83	0.222	4	2.2*
0.19	17.57	0.350	4	30.7*

* Effect was statistically significant (p≤0.05)

 

 

Mean body weight and percentage of reduction at the end of exposure

Conc. (mg solid content/L)	Mean	Std. Dev.	n	% Reduction
Control	158.250	12.0934	4
0.0082	147.250	8.1803	4	7.0*
0.020	143.500	3.8730	4	9.3*
0.072	144.500	6.2450	4	8.7*
0.19	55.000	3.1623	4	65.2*

* Effect was statistically significant (p≤0.05)

 

Effect parameters (mg solid content/L)

Parameter		NOEC	LOEC	EC10	EC50
Embryonic survival	Value^1	0.072	0.19	1.0	>1.0
Post-hatch survival	Value^2	0.072	0.19	0.097	0.20
	Lower 95% CI			0.075	0.17
	Upper 95% CI			0.12	0.24
Growth (length)	Value^2	0.0082	0.020	0.12	0.26
	Lower 95% CI			0.11	0.25
	Upper 95% CI			0.12	0.28
Growth (weight)	Value^2	<0.0082	0.0082	0.075	0.15
	Lower 95% CI			0.068	0.15
	Upper 95% CI			0.081	0.16

¹for day 5 of exposure,²for day 32 of exposure, cl–confidence limit, * - no confidence intervals could be given

Validity criteria fulfilled:

yes

Conclusions:

The NOEC, LOEC and EC10 for hatching were 0.072, 0.19 and 1.0 mg solid content/L, respectively.
The NOEC, LOEC and EC10 for post-hatch survival were 0.072, 0.19 and 0.097 mg solid content/L, respectively.
The NOEC, LOEC and EC10 for larval growth (length) were 0.0082, 0.020 and 0.12 mg solid content/L, respectively.
The NOEC, LOEC and EC10 for larval growth (weight) were <0.0082, 0.0082 and 0.075 mg solid content/L, respectively.

Executive summary:

The lethal and sub-lethal effects of 1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-,N-(C8-18(even numbered) acyl) derivs., hydroxides, inner salts on the early life stages of the freshwater fish,Pimephales promelas, was assessed according to the OECD Guideline 210 (Fish, Early-life Stage Toxicity Test). Test organisms were exposed to mean measured test concentrations of 0 (control), 0.0082, 0.020, 0.072, 0.19 and 1.0 mg solid content/L over a 32-day period. All of the prescribed guideline validity criteria were met and the study is considered to be valid and reliable.

 

The NOEC, LOEC and EC10 for hatching were 0.072, 0.19 and 1.0 mg solid content/L, respectively. The NOEC, LOEC and EC10 for post-hatch survival were 0.072, 0.19 and 0.097 mg solid content/L, respectively. The NOEC, LOEC and EC10 for larval growth (length) were 0.0082, 0.020 and 0.12 mg solid content/L, respectively. The NOEC, LOEC and EC10 for larval growth (weight) were <0.0082, 0.0082 and 0.075 mg solid content/L, respectively.

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source substance, C8-C18 AAPHS, has the same functional groups and general composition as the target C12-14 substance. The main variable resides in the alkyl chain distribution.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical = C8-18 cocamidopropyl hydroxysultaine (EC 939-455-3).
Target chemical = C12-14 cocamidopropyl hydroxysultaine (EC 293-878-1).

3. ANALOGUE APPROACH JUSTIFICATION
Cf. attaced Read-Across Justification Document (§13 Assessment reports).

4. DATA MATRIX
Cf. attached Read-Across Justification Document (§13 Assessment reports).

Reason / purpose for cross-reference:

read-across source

Specific details on test material used for the study:

Aqueous solution at 36.3% w/w of active content (= surface-active fraction, i.e. alkylamidopropylhydroxysultaine) is being used as a read across for 1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-C12-14 acyl derivs., hydroxides, inner salts

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.072 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

number hatched

Duration:

32 d

Dose descriptor:

LOEC

Effect conc.:

0.019 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

number hatched

Duration:

32 d

Dose descriptor:

EC10

Effect conc.:

1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

number hatched

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.072 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

other: Post-hatch survival

Duration:

32 d

Dose descriptor:

LOEC

Effect conc.:

0.019 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

other: Post-hatch survival

Duration:

32 d

Dose descriptor:

EC10

Effect conc.:

0.097 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

other: Post-hatch survival

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.008 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

length

Duration:

32 d

Dose descriptor:

LOEC

Effect conc.:

0.02 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

length

Duration:

32 d

Dose descriptor:

EC10

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

length

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

< 0.008 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

weight

Duration:

32 d

Dose descriptor:

LOEC

Effect conc.:

0.008 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

weight

Key result

Duration:

32 d

Dose descriptor:

EC10

Effect conc.:

0.075 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

solid content

Basis for effect:

weight

Details on results:

Measured Concentrations:
Analyses of samples taken from the nominal concentrations on a weekly basis showed that measured concentrations were generally at or below nominal (44-116%). Measurement of concentrations in the mixing flasks on Day 28 and in the stock on Day-1 showed that the concentrations were in agreement with nominal, i.e. 98-110% in the mixing flasks and 109-116% in the stock. It was therefore expected that the lower concentrations in the test vessels was related to the presence of fish. Following a worst-case scenario, the effect parameters were based on mean measured concentrations.

Embryonic survival and hatching:
The overall survival of embryos at the end of hatching was 98% for the control, which complies with the requirements of the guideline (i.e. at least 70% of the embryos in the control should hatch). Hatching success was 98-100% in concentrations up to and including 0.072 mg solid content/L and not statistically different from the control. Hatching success at the two highest concentrations of 0.19 and 1.0 mg solid content/L was 93 and 90%, respectively, but proved to be statistically different from the control. The test item caused earlier onset of hatching. First larvae were observed on day three of exposure and were mainly related to the two highest concentrations tested (i.e. 25 and 31%, of the introduced embryos, respectively, had hatched). On day four the percentage of hatching in all five test concentrations was 9, 29, 49, 71 and 86%, while hatching in the control did not start until day 5. Hatching was complete for the control and all test concentrations on day five of exposure.

Larval survival and development:
The mean post-hatch larval survival was 96% in the control. The validity criterion for posthatch survival of at least 75% was met. The survival rates ranged from 0 to ≥96% and were clearly dose-related. Statistical analyses indicated that larval survival was not statistically different from the control at test concentrations up to and including 0.072 mg solid content/L. Statistical analysis was not performed on abnormalities but based on the observations made during the test it appears that the severity and incidence of abnormalities were concentration dependent.

Effects on Larval Growth:
A statistically significant reduction of length was observed at concentrations of 0.020 mg solid content/L and higher, while reduction of weight was statistically significant at all concentrations tested.

All of the prescribed guideline validity criteria were met. Dissolved oxygen concentration was maintained above 60% of the air saturation value throughout the test. Water temperature did not differ by more than ± 1.5°C between test chambers between successive days at any time during the test, and was within the temperature ranges specified for the test species. Exposure concentrations during the test period were analytically monitored and effect parameters were based on mean measured concentrations. Overall survival of fertilised eggs in the control was >70% until hatching was complete (i.e. 98%). The overall survival of hatched larvae was >75% in the control during the remaining test period (i.e. 96%).

Reported statistics and error estimates:

The following statistical procedures were used to determine the NOEC and LOEC for hatching success, post-hatch survival, body weight and body length.

Hatching success and Post-hatch survival: Step-down Cochran-Armitage Test Procedure (α=0.05, one-sided greater).
Body weight and body length:
- Data distribution: Shapiro-Wilk´s Test
- Homogeneity of variance: Levene´s Test (with Residuals)
- Differences between treatments and the pooled control: Williams Multiple Sequential t-test Procedure (α=0.05, one-sided smaller).

Calculation of ECx-values was based on probit analysis using linear maximum likelihood regression with the percentages of post-hatch mortality and the percentages of reduction of growth (both weight and length) versus the logarithms of the corresponding average exposure concentrations of the test item.

All analyses were performed with ToxRat Professional 3.2.1 (ToxRat Solutions® GmbH, Germany).

Nominal, measured and mean measured concentrations during the ELS test

Nominal concentration Test item (mg solid content/L)	Measured concentration (mg solid content/L)								Mean conc. Test item (mg solid content/L)
	Day 0	Day^14	Day^15	Day 7	Day 14	Day 21	Day 28	Day 32
0.010	0.0116	n.m.	n.m.	0.0063	0.00898	0.0074	0.0068	0.0079	0.0082
0.032	0.0337	n.m.	n.m.	0.0181	0.0190	0.0151	0.0141	0.0192	0.020
0.10	0.108	n.m.	n.m.	0.0668	0.0765	0.0685	0.0578	0.0526	0.072
0.32	0.336	0.00873	0.000047	0.254	0.278	0.257	0.220	0.205	0.19
1.0	1.09	n.m.	n.m.	0.920	n.m.	n.m.	n.m.	n.m.	1.0

n.m. Not measured

¹Samples taken after a dosing failure that lasted for 16 hours on Day 4 and for at least 1¼ hours on Day 5

 

 

Post-Hatch Survival at the End of Exposure (Day 32)

Conc. (mg solid content/L)	Total Hatched	Survived	Dead	% Post-hatch Mortality
Control	78	75	3	3.8
0.0082	78	76	2	2.6
0.020	80	77	3	3.8
0.072	78	75	3	3.8
0.19	74	40	34	45.9*
1.0	72	0	72	100.0*

* Effect was statistically significant (p≤0.05)

 

 

Mean body length (mm) and percentage of reduction at the end of exposure

Conc. (mg solid content/L)	Mean	Std. Dev.	n	% Reduction
Control	25.38	0.386	4
0.0082	25.08	0.369	4	1.2
0.020	24.80	0.258	4	2.3*
0.072	24.83	0.222	4	2.2*
0.19	17.57	0.350	4	30.7*

* Effect was statistically significant (p≤0.05)

 

 

Mean body weight and percentage of reduction at the end of exposure

Conc. (mg solid content/L)	Mean	Std. Dev.	n	% Reduction
Control	158.250	12.0934	4
0.0082	147.250	8.1803	4	7.0*
0.020	143.500	3.8730	4	9.3*
0.072	144.500	6.2450	4	8.7*
0.19	55.000	3.1623	4	65.2*

* Effect was statistically significant (p≤0.05)

 

Effect parameters (mg solid content/L)

Parameter		NOEC	LOEC	EC10	EC50
Embryonic survival	Value^1	0.072	0.19	1.0	>1.0
Post-hatch survival	Value^2	0.072	0.19	0.097	0.20
	Lower 95% CI			0.075	0.17
	Upper 95% CI			0.12	0.24
Growth (length)	Value^2	0.0082	0.020	0.12	0.26
	Lower 95% CI			0.11	0.25
	Upper 95% CI			0.12	0.28
Growth (weight)	Value^2	<0.0082	0.0082	0.075	0.15
	Lower 95% CI			0.068	0.15
	Upper 95% CI			0.081	0.16

¹for day 5 of exposure,²for day 32 of exposure, cl–confidence limit, * - no confidence intervals could be given

Validity criteria fulfilled:

yes

Conclusions:

The NOEC, LOEC and EC10 for hatching were 0.072, 0.19 and 1.0 mg solid content/L, respectively.
The NOEC, LOEC and EC10 for post-hatch survival were 0.072, 0.19 and 0.097 mg solid content/L, respectively.
The NOEC, LOEC and EC10 for larval growth (length) were 0.0082, 0.020 and 0.12 mg solid content/L, respectively.
The NOEC, LOEC and EC10 for larval growth (weight) were <0.0082, 0.0082 and 0.075 mg solid content/L, respectively.

Executive summary:

The chronic effects of C12-14 cocamidopropyl hydroxysultaine (C12-14 AAPHS) on the early life stages of the freshwater fish species, Pimephales promelas, have been estimated based on read-across from a study testing C8-18 cocamidopropyl hydroxysultaine (C8-18 AAPHS). Given that C12-14 AAPHS and C8-18 AAPHS share the same functional groups and general composition, the main variable being alkyl chain distribution, C8-18 AAPHS was considered appropriate for the purposes of read-across to C12 -14 AAPHS.

The lethal and sub-lethal effects of the test item on the early life stages of P. promelas, was assessed according to the OECD Guideline 210 (Fish, Early-life Stage Toxicity Test). Test organisms were exposed to mean measured test concentrations of 0 (control), 0.0082, 0.020, 0.072, 0.19 and 1.0 mg solid content/L over a 32-day period. All of the prescribed guideline validity criteria were met and the study is considered to be valid and reliable.

 

The NOEC, LOEC and EC10 for hatching were 0.072, 0.19 and 1.0 mg solid content/L, respectively. The NOEC, LOEC and EC10 for post-hatch survival were 0.072, 0.19 and 0.097 mg solid content/L, respectively. The NOEC, LOEC and EC10 for larval growth (length) were 0.0082, 0.020 and 0.12 mg solid content/L, respectively. The NOEC, LOEC and EC10 for larval growth (weight) were <0.0082, 0.0082 and 0.075 mg solid content/L, respectively.

 

C8 -18 AAPHS is considered appropriate for read-across purposes to C12 -14 AAPHS.

Description of key information

The chronic effects of C12-14 cocamidopropyl hydroxysultaine (C12-14 AAPHS) on the early life stages of the freshwater fish species, Pimephales promelas, have been estimated based on read-across from a reliable study testing C8-18 cocamidopropyl hydroxysultaine (C8-18 AAPHS) according to the OECD Guideline 210 (Fish, Early-life Stage Toxicity Test). Given that C12-14 AAPHS and C8-18 AAPHS share the same functional groups and general composition, the main variable being alkyl chain distribution, C8-18 AAPHS was considered appropriate for the purposes of read-across to C12 -14 AAPHS.

Test organisms were exposed to the test item over a 32-day period. Effect concentrations were based on mean measured concentrations. The NOEC, LOEC and EC10 for hatching were 0.072, 0.19 and 1.0 mg solid content/L, respectively. The NOEC, LOEC and EC10 for post-hatch survival were 0.072, 0.19 and 0.097 mg solid content/L, respectively. The NOEC, LOEC and EC10 for larval growth (length) were 0.0082, 0.020 and 0.12 mg solid content/L, respectively. The NOEC, LOEC and EC10 for larval growth (weight) were <0.0082, 0.0082 and 0.075 mg solid content/L, respectively.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

EC10

Effect concentration:

0.075 mg/L