(8α,9R,8'''α,9'''R)-1,1'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(methylene)]bis(6'-methoxycinchonan-1-ium-9-ol) dibromide

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date: October 03, 2019 Experimental start date: October 03, 2019 Experimental completion date: December 04, 2019 Study completion date: January 29, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD 203 and under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss GLP Monitoring Authorities, Date of decission 2018-01-10

Specific details on test material used for the study:

Appearance: Beige solid
Expiration Date: End of February 2021
Storage Conditions: < 30 °C

Analytical monitoring:

yes

Details on sampling:

- Sampled concentrations: 6.25, 12.5, 25, 50, and 100 mg/L
- Sampling method: For the determination of the test item CA5727A concentrations, duplicate samples
were taken from the test media of all test concentration groups and the control at the start and at the
end of each test medium renewal period. However, for the highest nominal test concentration of
100 mg/L, last samples were taken at 0 hours as 100 % mortality was observed after 2 hours of
exposure time.
- Sample storage conditions before analysis: all samples were frozen (at -20 ± 5 °C)
- Analysed samples: The concentrations of the test item CA5727A were measured in one of the
duplicates for all taken samples.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: At the start of the test (Day 0) and at the test medium renewal after 48 hours test duration
(Day 2), a stock solution with a nominal test item concentration of 500 mg/L was prepared by mixing
2.501 g test item into 5000 mL test water on Day 0 and 1.500 g test item into 3000 mL test water on
Day 2 using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature.
- Eluate:This intensively mixed stock solution was used in a series of dilutions to prepare the test
media of all test item concentrations. The test media were freshly prepared just before introduction
of the fish (at the start of the exposure and before test media renewal).
For each treatment 12 liters of test medium were used for exposure of the fish.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No remarkable observations
were made concerning the appearance of the test media.
All test media were clear solutions throughout the entire test duration.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout (Oncorhynchus mykiss)
- Source:The fish were obtained from a commercial breeder at least 12 days before start of the test
(fish breeding farm Störk, 88348 Bad Saulgau, Germany).
- Length at study initiation (length definition, mean, range and SD): The mean total body length
(measured from the mouth to the end of the tail fin) of the fish was 3.4 ± 0.25 cm (Mean ± SD)
- Weight at study initiation (mean and range, SD): The mean body wet weight was 0.35 ± 0.10 g
(Mean ± SD).

ACCLIMATION
- Acclimation period: During the 12 days prior to the main test, 0.7 % (1 of 150 fish) mortality
was observed in the test fish batch and all surviving fish were healthy. No medication was
applied during holding and acclimatization of the fish.
- Acclimation conditions (same as test or not):same as test
- Type and amount of food during acclimation: During holding and acclimatization until one
day before the start of the test, the fish were fed with a commercial fish diet (Hokovit,
Hofmann Nutrition AG, CH-4922, Bützberg, Switzerland).
- Health during acclimation (any mortality observed): During the 12 days prior to the main
test, 0.7 % (1 of 150 fish) mortality was observed in the test fish batch and all surviving
fish were healthy. No medication was applied during holding and acclimatization of the fish.

FEEDING DURING TEST
No feeding during the test

Test type:

semi-static

Water media type:

other: Reconstituted test water (EPA Medium) was used in the study

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

A semi-static test design with a medium renewal after 48 hours was applied to keep the test item concentrations in the test media as constant as possible during the test period of 96 hours.

Hardness:

120 mg CaCO3/L

Test temperature:

13 °C

pH:

7.0-7.2

Dissolved oxygen:

9.4-9.8 mg/L

Conductivity:

The conductivity was 353 μS/cm

Nominal and measured concentrations:

nominal concentrations (measured concentrations 0/48h first renewal period): 6.25 (6.0/6.1),
12.5 (12/12), 25 (24/23), 50 (49/48) and 100 (93*) mg/L (*for the highest nominal test concentration
of 100 mg/L, last samples were taken at 0 hours as 100 % mortality was observed after 2 hours
of exposure time nominal concentrations (measured concentrations 0/48h second renewal period):
6.25 (5.8/5.9), 12.5 (12/11), 25 (23/23), 50 (46/45)

Details on test conditions:

TEST SYSTEM
- Test vessel: 13 liter mono block glass aquarium with 12 liters test medium
- Type (delete if not applicable): open
- Aeration: no
- Renewal rate of test solution (frequency): 48 hours
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.20 g fish wet weight per liter medium

TEST MEDIUM / WATER PARAMETERS
-Ingredients / Properties [mmol/L] / Concentration [mg/L]
CaCl2 × 2H2O / 1.0 / 147
MgSO4 × 7H2O / 0.25 / 61.5
NaHCO3 / 0.38 / 32.5
KCl / 0.038 / 2.9
- Alkalinity:0.5 mmol/L

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod:16-hour light to 8-hour dark cycle with a 30-minute transition period
- Light intensity:12 and 14 µE m-2 s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The test fish were
observed for mortality and visible abnormalities approximately 2 and 5 hours after the start of the
exposure and further on each exposure day in the morning and the afternoon.
The fish were checked for the mandatory clinical signs presented below, including the
corresponding sub-categories (as presented in Annex 4 of the test guideline):
E: Loss of equilibrium
S: Abnormal swimming behavior
V: Ventilatory function
P: Abnormal skin pigmentation
O: Other visible abnormalities

NOEC and LOEC were determined from the raw data based on the occurrence of mortality.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 1.0 / 10 / 100 mg/L
- Results used to determine the conditions for the definitive study:
96-hour mortality: 1.0 and 10 mg/L / 0%, 100 mg/L /100%

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

71 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: In the control and the test item concentrations up to and including
nominal 25 mg/L all fish survived until the end of the test and no visible abnormalities were
observed in the test fish. At the next higher test concentration of 50 mg/L all fish survived
until the end of the test, but all test fish showed several visible abnormalities during the
exposure phase. At the highest test concentration of 100 mg/L all fish were dead after two
hours of the exposure
- Mortality of control: no
- Other adverse effects control: no

Reported statistics and error estimates:

The LC50 at the observation intervals after 2, 5, 24, 48, 72 and 96 hours could not be calculated by
Probit Analysis or Moving Average Interpolation due to the steep concentration-effect relationship.
Instead, the LC50-values and the 95 % confidence limits were estimated using the binominal distribution.
For the calculation of the LC50 values, the results from the observations in the afternoon were used,
since these are the time points where the 24, 48, 72 and 96-hour observation intervals were completed.

Sublethal observations / clinical signs:

Biological Results: Cumulative Mortality and Visible Abnormalities [Number of Fish]

			Day 0	Day 0	Day 1	Day 1		Day 2		Day 2		Day 3	Day 3	Day 4	Day 4
Nominal Concentration of CA5727A [mg/L]			2 hrs after start	5 hrs after start	a.m.	p.m.		a.m.		p.m.		a.m.	p.m.	a.m.	p.m.
Control	M		0	0	0	0		0		0		0	0	0	0
Control	VA		0	0	0	0		0		0		0	0	0	0
6.25	M		0	0	0	0		0		0		0	0	0	0
	VA		0	0	0	0		0		0		0	0	0	0
12.5	M		0	0	0	0		0		0		0	0	0	0
	VA		0	0	0	0		0		0		0	0	0	0
25	M		0	0	0	0		0		0		0	0	0	0
	VA		0	0	0	0		0		0		0	0	0	0
50	M		0	0	0	0		0		0		0	0	0	0
	VA		7S	7S	7S	7S		7S		7S;1E		7S;3E	7S;3E	7S;3E	7S;4E
100	M		7	7	7	7		7		7		7	7	7	7
	VA		--	--	--	--		--		--		--	--	--	--
LC50**			71	71	71		71		71		71	71	71	71	71
95 % C.I.			50-100	50-100	50-100		50-100		50-100		50-100	50-100	50-100	50-100	50-100

Analytical Results

Sampling Day/ Sample Age	Nominal Concentration of Test Item	Determined Concentration of Test item	% of Nominal Concentration
[day/hours]	[mg/L]	[mg/L]	[%]
0/0	Control	n.d.	-
	6.25	5.96	95
	12.5	11.9	95
	25	23.5	94
	50	49.1	98
	100	92.5	93
2/48	Control	n.d.	-
	6.25	6.07	97
	12.5	11.9	95
	25	23.4	94
	50	48.4	97
2/0	Control	n.d.	-
	6.25	5.80	93
	12.5	12.1	97
	25	23.4	94
	50	46.1	92
4/48	Control	n.d.	-
	6.25	5.94	95
	12.5	11.3	91
	25	23.3	93
	50	44.9	90

 

Validity criteria fulfilled:

yes

Conclusions:

The substance had adverse effects to freshwater fish (rainbow trout) in an acute toxicity study. The 96-hour LC50 = 71 mg/L.

Executive summary:

The acute toxicity of the test item to juvenile fish of the speciesrainbow trout (Oncorhynchus mykiss) was determined in a 96‑hour semi-static test according to the OECD Guideline for the Testing of Chemicals, No. 203, Fish, Acute Toxicity Test, 2019. The following nominal concentrations of the test item were tested: 6.25, 12.5, 25, 50 and 100 mg/L. A control (test water without test item) was tested in parallel. A semi-static test design with a medium renewal after 48 hours was applied.

The measured concentrations of the test item in the test media ranged between 92 and 98 % of the nominal values at the start of the renewal periods and between 90 and 97 % of nominal at the end of both test medium renewal periods. Thus, the analytical results confirm the correct dosage and stability of the test item over the exposure period. Therefore, the endpoint values were reported on the nominal concentrations of the test item. Seven fish were used per treatment. 

The fish were observed for visible abnormalities and mortality at 2 and 5 hours after test start and twice every 24 hours period thereafter. 

Description of key information

96-hour LC50 = 71 mg/L (Onchorhynchus mykiss, OECD TG 203, GLP)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

71 mg/L