Quaternary ammonium compounds, bis(hydrogenated tallow alkyl)dimethyl, chlorides, reaction products with polyethylenepolyamines and tall-oil fatty acids, humates hydrochlorides

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:

15 Feb 2019 - 03 Jun 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No analytical verification of the test substance has been performed.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

Deviations:

yes

Remarks:

Analytical measurement methods of the chemical active ingredient cannot be performed by any modern analytical method

GLP compliance:

yes

Analytical monitoring:

no

Remarks:

The chemical active ingredient cannot be analyzed by any modern analytical methods based on information from the sponsor.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Limit water soluble fraction stock solution was prepared daily by dissolving 0.10 g of test substance in 100 mL of dilution water and mixing for approximately 23 hours, then allowing it to settle for 1 hour; dosing solution of 12 mg/L was prepared daily by combining 48 mL of the stock solution with 3952 mL of dilution water.
- Controls: dilution water only

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Source: STILLMEADOW, Inc.

POST-HATCH FEEDING
Newly hatched: Artemia nauplii: 1) minimally within 2 days of hatching and 2) ad libitum at least twice daily with minimal surplus after 90% of eggs have hatched
Juveniles: 48 hour-old Artemia nauplii and flake food ad libitum at least twice daily with minimal surplus.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

28 d

Hardness:

80 - 100 mg/L (as CaCO3)

Test temperature:

25 °C

pH:

7.4 - 8.2

Dissolved oxygen:

60 - 99%

Nominal and measured concentrations:

Nominal test concentrations: 0 (control), 12 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 1 L glass beaker
- Headspace: 100 mL, fill volume: 900 mL
- Renewal rate of test solution (frequency/flow rate): Solutions were renewed daily by siphoning old solutions out of the test containers and replacing them with new test solutions; larva remained in the containers along with approximately 20% of the old test solution.
- No. of fertilized eggs/embryos per vessel: 25
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: max. 5.0 g fish/L loading

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: moderately hard synthetic freshwater
- Culture medium different from test medium: no
- Intervals of water quality measurement: dissolved oxygen, pH, conductivity, temperature (daily)

OTHER TEST CONDITIONS
- Photoperiod: 16/8 h light/dark

EFFECT PARAMETERS MEASURED: mortality (daily), wet weight and individual total lengths (test end)

RANGE-FINDING STUDY
- Nominal test concentrations: 1.0, 10, 100, 500 and 1000 mg/L
- Results used to determine the conditions for the definitive study: NOECmortality (96 h): > 1000 mg/L

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: egg hatching success, weight

Duration:

27 d

Dose descriptor:

NOEC

Effect conc.:

>= 12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Final survival based on Day 27 counts as there were two fish lost (one from each group) on Day 28 due to technician error.

Details on results:

- Other biological observations: no abnormal observations during the larval phase of testing.
- Effect concentrations exceeding solubility of substance in test medium: test substance adhering to the eggs at tested concentration of 12 mg/L

Reported statistics and error estimates:

For survival, an unpaired t-test was conducted. For length and weights, an unpaired t-test with transformed data was conducted. For the number of hatched eggs and the percent of hatched eggs, an ANOVA was conducted. Analysis was conducted using GraphPad InStat version 3.06 for Windows 95, GraphPad Software, San Diego California USA.

Table 1: 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).	25°C throughout the test	yes
The analytical measure of the test concentrations is compulsory.	Mesurement not possible	not applicable
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 > 70% Post-hatch success > 75%	yes

Table 2: egg hatching summary

Replicate	Beginning number of Eggs	Number Hatched
		Hours
Control	0 Hour	24	48	72	96	120
A	25	3	6	16	24	25
B	25	1	7	15	20	18
C	25	1	6	14	22	25
D	25	2	4	10	18	18
Total	100					86
12 mg/L
A	25	2	9	16	25	24
B	25	0	3	7	10	9
C	25	4	10	17	18	18
D	25	0	2	8	18	19
Total	100					70

Table 3: Limit test mortality results

Total Number of Surviving Juveniles for All Replicates
	Day 0 a	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7
Control	86	83	83	81	81	80	78	78
12 mg/L	70	70	69	67	67	67	67	65
	Day 8	Day 9	Day 10	Day 11	Day 12	Day 13	Day 14	Day 15
Control	78	75	74	74	74	74	74	74
12 mg/L	65	65	65	65	65	65	65	64
	Day 16	Day 17	Day 18	Day 19	Day 20	Day 21	Day 22	Day 23
Control	74	74	74	72	72	72	72	72
12 mg/L	64	64	64	63	63	63	63	63
	Day 24	Day 25	Day 26	Day 27	Day 28	Final Survival (%) b
Control	71	71	71	71	70		82.6%
12 mg/L	63	63	63	63	62		90.0%

a: Beginning number of juveniles from the egg hatching phase.

b: Final survival based on Day 27 counts as there were two fish lost (one from each group) on Day 28 due to technician error.

Table 4: Final measurement summary

Group/Replicate	Wet Weight (g)	Length(cm)	Group/Replicate	Wet Weight (g)	Length (cm)
Control			12mg/L
A	0.0871	1.5	A	0.1094	1.7
B	0.0801	1.6	B	0.2294	2.6
C	0.0690	1.7	C	0.0948	1.9
D	0.0941	1.8	D	0.0971	1.9
Mean	0.0826	1.6	Mean	0.1327	2.0
SD	0.0107	0.1	SD	0.0648	0.4
SE	0.0053	0.1	SE	0.0324	0.2

Test results of the additional test: The additional control resulted in a hatching success of 87%. The filtered and unfiltered hatching success rates were 69% and 61%, respectively. In the unfiltered trial, some test material was stuck to the eggs and some fish that were found dead were either hatched or half-hatched. In the filtered trial, no test material was stuck to the eggs but in replicate B, some eggs were dead that were half-hatched out.

Table 5: Additional egg trial results on hatching success

Replicate	Beginning number ofEggs		NumberHatched
				Hours
Control	0 Hour	24	48	72	96	120
A	25	0	4	6	21	23
B	25	0	0	3	17	22
C	25	0	1	9	19	18
D	25	0	0	12	20	24
Total	100					87
12 mg/L - Filtered
A	25	0	0	6	19	21
B	25	0	4	7	11	8
C	25	0	0	6	17	17
D	25	0	2	21	23	23
Total	100					69
12 mg/L - Unfiltered
A	25	0	0	4	19	18
B	25	0	3	3	14	13
C	25	0	5	2	17	17
D	25	0	2	5	15	13
Total	100					61

Validity criteria fulfilled:

yes

Remarks:

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

Additional information

One experimental GLP study is available testing the long-term toxicity of the test substance towards fish. This 28-day limit test was conducted with the frehwater species Pimephales promelas in a semi-static system according to OECD 210. Four replicates of 25 eggs each were exposed to the limit test substance concentration of 12 mg/L and moderately hard synthetic freshwater only (negative control), respectively. After hatching, the surviving juveniles were observed daily. Furthermore, three additional egg trials were conducted in parallell since it was observed that test substance adhered to the eggs. An additional control, a test group with filtered test substance (12 mg/L) and a test group with unfiltered test substance (12 mg/L) were tested to check if test material was sticking to the eggs and if hatching success or larval survival were negatively affected by the test substance.

For the control and test group (at 12 mg/L) in the main test, the hatching success was 86.0% and 71.0%, respectively and the percent of juvenile survival was 82.6% and 90.0%, respectively.

In the unfiltered trial of the additional test, some test material was stuck to the eggs. In the filtered trial, no test material was stuck to the eggs but in one replicate, some eggs were dead that were half-hatched out. The additional control resulted in a hatching success of 87%. The filtered and unfiltered hatching success rates were 69% and 61%, respectively.

There was no statistically significant differences for egg hatching success, juvenile survival, or final weights between the control and test groups in the main test. Furthermore, no significant difference in the mean hatching success from any group (test group, filtered group, unfiltered group and additional control group) compared to the original control was detected. Therefore, the NOELR for hatching success, juvenile success, and growth (as weight gain) was ≥

12 mg/L (nominal).