(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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date: September 02, 2019; Experimental start date: September 10, 2019; Experimental completion date: December 03, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

adopted 2006, corrected 2011

Deviations:

no

Remarks:

96 h exposure duration

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:

Sampling
- Sampled concentrations:All test concentrations
- Sampling method: duplicate samples (10 mL)
were taken from the test media of all test concentration groups and the control at the start and at the end of the test (96 hours). For sampling at the end of the test, the contents of the replicate test vessels were combined prior to sampling. As the test item was unstable, additional stability samples were taken after 48 hours following the recommendation of the test guideline. The analytical stability samples (10 mL sample volume) at 48 hours could not be taken from the test vessels itself due to the limited test medium volume (30 mL). Therefore, for each of these sampling dates, a set of additional flasks containing the corresponding test medium (with algae) was incubated under conditions identical to the test.
- Sample storage conditions before analysis: all samples were frozen (at -20 ± 5 °C)
- Analysed samples: The concentrations of the test item were measured in at least 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 a stock solution with the nominal test item concentration of 32 mg/L was prepared by mixing 16.04 mg of the test item into 500 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature. This intensively mixed stock solution was used to prepare an intermediate dilution with a nominal test item concentration of 3.2 mg/L. This second stock solution was diluted with test water to prepare the test medium of the highest test (nominal) concentration of 320 μg/L. All lower test concentrations were prepared in a series of dilutions
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
- 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 test period.
- Other relevant information: None

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Test type:

static

Water media type:

freshwater

Remarks:

Reconstituted test water (AAP Medium) prepared according to the test guideline was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water.

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

In agreement with the Test Guideline (TG) the normal exposure duration of 72 h was extended to 96 h because all validity criteria of in paragraph 11 of the TG could be met.

Post exposure observation period:

Not performed

Hardness:

The calculated water hardness of the test water was 0.15 mmol/L (= 15 mg/L as CaCO3).

Test temperature:

The temperature during the test was maintained at 21 °C.

pH:

The pH in the test water (AAP medium) was 7.5.
The pH of the test media was in the range of 7.4 to 8.6 during the test period.
The pH rose in the control from 7.4 at test start to 8.1 at test end fulfilling the requirement of the OECD guideline that the pH of the control medium should not increase by more than 1.5 units during the test.

Dissolved oxygen:

Not determined.

Salinity:

Not determined.

Conductivity:

Not determined.

Nominal and measured concentrations:

Nominal test item concentrations tested were 1.0, 3.2, 10, 32, 100 and 320 μg/L.
Geometric Mean Measured concentrations were 0.22, 0.30, 1.5, 2.9, 34 and 222 μg/L.
The measured concentrations of CA5727A in the test media of all test concentrations were between 64 and 93 % of the nominal values at the start of the test. After 48 hours of the exposure, the test item concentrations in the test media were between Due to the decrease of test item concentrations during the test period, the mean measured test item concentrations were calculated as the time-weighted geometric means over all measurements, with the exception of the nominal test item concentration of 10 μg/L. At this concentration the mean measured concentration was calculated as the geometric mean of the concentrations measured at the start and end of the test, since the measurement after 48 hours was

Details on test conditions:

DETAILS ON TEST CONDITIONS
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glas, 75 mL, open, 30 mL
- Aeration: No
- Initial cells density: 5000 cells/mL (corresponding 0.61E+04 relative fluorescence)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, AAP-Medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Macro-nutrients: Ingredients / Concentration
NaHCO3 / 15.0 mg/L
K2HPO4 / 1.044 mg/L
MgSO4 × 7 H2O / 14.6 mg/L
MgCl2 × 6 H2O / 12.16 mg/L
CaCl2 × 2 H2O / 4.41 mg/L
NaNO3 / 25.5 mg/L
Trace elements: Ingredients / Concentration
H3BO3 / 186.0 µg/L
MnCl2 × 4 H2O / 415.0 µg/L
ZnCl2 / 3.27 µg/L
CoCl2 × 6 H2O / 1.43 µg/L
CuCl2 × 2 H2O / 0.012 µg/L
Na2MoO4 × 2 H2O / 7.26 µg/L
FeCl3 × 6 H2O / 160.0 µg/L
Na2EDTA × 2 H2O: 300.0 µg/L

- CaCO3: 15 mg/L
- Water hardness: 0.15 mmol/L (= 15 mg/L as CaCO3)
- Culture medium different from test medium: No
- Intervals of water quality measurement: it is a reconstituted test water

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: No
- Photoperiod: continuously illuminated
- Light intensity and quality: ca. 75 µE/sm² (range: 73 to 76), LED measured at nine places in the experimental area). The light intensity over the incubation area was within a ±15 %-deviation from the average light intensity as
recommended by the guideline.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of biomass: fluorimeter (0, 24, 48, 72 and 96 hours). (For the inoculum culture: electronic particle counter for determination of cell concentration)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2

RANGE-FINDING STUDY
- Test concentrations: 0.1 / 1.0 / 10 / 100 μg/L and 3.2, 32 and 320 μg/L
- Results used to determine the conditions for the definitive study:
Concentration / Inhibition of Average Growth after 72 hours / Inhibition of Yield after 72 hours
3.2μg/L / -2.0 % / -11 %
32 μg/L / -20 % / -70 %
320 μg/L / -87 % / -100 %

Reference substance (positive control):

yes

Remarks:

Results of the reference test for potassium dichromate (CAS 7778-50-9) confirmed sensitivity of the test system (IES Study 20190094, 2019-05-19).

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.7 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 2.0 – 3.4 µg/L; NOEC 1.5 µg/L (Williams t-test, one-sided smaller, α = 0.05); LOEC 2.9 µg/L.

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

7.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 2.5 – 12 µg/L; NOEC 1.5 µg/L (Williams t-test, one-sided smaller, α = 0.05); LOEC 2.9 µg/L.

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

5.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 4.8 – 7.0 µg/L

Duration:

96 h

Dose descriptor:

EC20

Effect conc.:

13 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 5.9 – 18 µg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

27 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 23 – 30 µg/L

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

35 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 26 – 44 µg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI 0.94 – 1.4 µg/L

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

1.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI 0.94 – 1.4 µg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI 1.4 – 2.0 µg/L

Duration:

96 h

Dose descriptor:

EC20

Effect conc.:

1.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI 1.7 – 2.1 µg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI 3.5 – 5.2 µg/L

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

4.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI 4.2 – 5.8 µg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Calculated as Area Under Curve (AUC)

Remarks on result:

other: 95% CI 1.2 – 1.7 µg/L; NOEC 0.3 µg/L (Welch t-test with Bonferroni-Holm-adjustment, one-sided smaller, α = 0.05); LOEC 1.5 µg/L.

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

1.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Calculated as Area Under Curve (AUC)

Remarks on result:

other: 95% CI 1.0 – 1.5 µg/L; NOEC 0.3 µg/L (Welch t-test with Bonferroni-Holm-adjustment, one-sided smaller, α = 0.05); LOEC 1.5 µg/L.

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.9

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Calculated as Area Under Curve (AUC)

Remarks on result:

other: 95% CI 1.7 – 2.1 µg/L

Duration:

96 h

Dose descriptor:

EC20

Effect conc.:

1.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Calculated as Area Under Curve (AUC)

Remarks on result:

other: 95% CI 1.7 – 2.1 µg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Calculated as Area Under Curve (AUC)

Remarks on result:

other: 95% CI 2.9 – 3.7 µg/L

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

3.9

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Calculated as Area Under Curve (AUC)

Remarks on result:

other: 95% CI 3.5 – 4.7 µg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the mean measured concentration of 34 μg/L (100 μg/L nominal) and the algal cells in the control. The shape and size of the algal cells were not affected by the test item at any of the test concentrations tested.
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: None
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: NO

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 72-hour EC50 for growth rate: 1.1 mg potassium dichromate/L with 95 % confidence limits of 1.0 and 1.1 mg/L.

Reported statistics and error estimates:

Growth rate and yield were calculated for each test flask. The mean values for growth rate and yield were calculated for each treatment.
The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95 % confidence intervals were calculated by Probit Analysis using linear maximum likelihood regression.
For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-test and Welch t-test with Bonferroni-Holm-adjustment, where appropriate.

The measured concentrations of the test item CA5727A in the test media of all test concentrations at the start, after 48 hours and at the end of the test are shown in the table below.

 

Nominal Test Item Concentration [µg/L]	Analytically Measured Concentration of the Test Item [µg/L]			Mean Measured Concentration (Geometric Mean*) [µg/L]
	0 Hours	48 Hours	96 Hours
1.0	0.664	<LOQ	< LOQ	0.22
3.2	2.27	<LOQ	< LOQ	0.30
10	7.77	<LOQ**	0.309	1.5^$
32	26.4	1.23	1.68	2.9
100	88.2	29.7	17.2	34
320	299	198	207	222

 

LOQ:     Limit of quantification (LOQ = 0.299 µg test item/L).

*:           If the measured concentration was below LOQ, half of the LOQ (½ LOQ = 0.150 µg/L) was used to calculate the mean measured concentration.

**:        The measurement (<LOQ) after 48 hours was not considered in the calculation of the mean measured concentration, since in the 96-hour samples test item above the LOQ was determined.

$:          This mean measured concentration was calculated as the geometric mean of the concentrations measured at the start and end of the test.

Average Growth Rates

Nominal Test Item Concentration     [µg/L]	Mean Measured Test Item Concentration [µg/L]	Repl. No.	Average Growth Rate µ [day^-1]
			24 Hours	48 Hours	72 Hours	96 Hours
Control		1	1.57	1.52	1.59	1.51
		2	1.14	1.56	1.57	1.50
		3	1.76	1.57	1.62	1.48
		4	1.21	1.54	1.59	1.49
		5	1.63	1.57	1.59	1.49
		6	1.37	1.54	1.59	1.47
		Mean	1.45	1.55	1.59	1.49
		SD	0.247	0.018	0.014	0.015
1.0	0.22	1	1.57	1.59	1.56	1.51
		2	1.56	1.55	1.55	1.51
		3	1.36	1.67	1.63	1.51
		Mean	1.50	1.60	1.58	1.51
		SD	0.121	0.061	0.039	0.002
3.2	0.30	1	1.27	1.58	1.56	1.50
		2	1.59	1.60	1.59	1.50
		3	1.50	1.58	1.58	1.50
		Mean	1.45	1.59	1.58	1.50
		SD	0.167	0.013	0.019	0.005
10	1.5	1	1.24	1.56	1.56	1.48
		2	1.60	1.54	1.55	1.45
		3	1.27	1.53	1.57	1.44
		Mean	1.37	1.54	1.56	1.46
		SD	0.199	0.013	0.012	0.023
32	2.9	1	1.26	0.901	1.44	1.40
		2	1.28	0.819	1.35	1.36
		3	1.26	1.40	1.45	1.40
		Mean	1.26	1.04	1.41	1.39
		SD	0.014	0.312	0.057	0.026
100	34	1	-0.662	0.141	0.754	0.758
		2	-0.692	0.461	0.636	0.795
		3	0.180	0.075	0.644	0.788
		Mean	-0.392	0.226	0.678	0.780
		SD	0.495	0.206	0.066	0.020
320	222	1	-0.475	-0.093	0.208	-0.044
		2	-0.244	0.110	0.325	0.145
		3	-0.313	-0.884	0.149	-0.418
		Mean	-0.344	-0.289	0.228	-0.105
		SD	0.119	0.525	0.090	0.287

SD:        Standard deviation

Inhibition of the Average Growth Rate

Nominal Test Item Concentration	Mean Measured Concentration	Average Growth Rate µ [day^-1] and Inhibition Ir[%]
		0-24 h		0-48 h		0-72 h		0-96 h
[µg/L]	[µg/L]	µ [day^-1]	Ir[%]	µ [day^-1]	Ir[%]	µ [day^-1]	Ir[%]	µ [day^-1]	Ir[%]
Control	---	1.45	0.0	1.55	0.0	1.59	0.0	1.49	0.0
1.0	0.22	1.50	-3.6	1.60	-3.5	1.58	0.6	1.51	-1.4
3.2	0.30	1.45	-0.6	1.59	-2.5	1.58	1.0	1.50	-0.7
10	1.5	1.37	5.1	1.54	0.4	1.56	2.1	1.46	2.3
32	2.9	1.26	13	1.04*	33	1.41*	11	1.39^#	7.0
100	34	-0.39*	127	0.23*	85	0.68*	57	0.78^#	48
320	222	-0.34*	124	-0.29*	119	0.23*	86	-0.11^#	107

 

*:        Mean value statistically significantly lower than in the control (according to a Williams t‑test one-sided smaller,a= 0.05).

#:        Mean value statistically significantly lower than in the control (according to a Welch t-test one-sided smaller,a= 0.05).

Note:  Percentage inhibition values in excess of 100 % are obtained when the biomass at the end of the interval is lower than at the start of the interval.

Validity criteria fulfilled:

yes

Remarks:

Biomass increased by factor of 118 (72h); cirterion ≥ 118 Mean CV daily growth rates (section-by-section) 14 % (72h) & 19 % (96h); criterion ≤ 35 % CV average specific growth rates in replicates was 1.3 % (72h); criterion ≤ 7%

Conclusions:

In a valid and reliable study the effect of the test item on the growth of the freshwater green algal species Raphidocelis subcapitata was ErC10(72h) 2.7 μg/L, ErC50(72h) 27 μg/L.

Executive summary:

The effect of the test item on the growth of freshwater green algae (Raphidocelis subcapitata) was investigated in a 96-hour static test according to the OECD Guideline 201 (2006, corrected 2011).

The nominal test item concentrations tested were 1.0, 3.2, 10, 32, 100 and 320 μg/L. A control (test water without test item) was tested in parallel.

The measured concentrations of the test item in the test media of all test concentrations were between 64 and 93% of the nominal values at the start of the test. After 48 hours of the exposure, the test item concentrations in the test media were between <LOQ (analytical limit of quantification, LOQ = 0.299 μg/L) and 62% of the nominal values. At the end of the test, the test item concentrations in the test media were between <LOQ and 65% of the nominal values. Due to the decrease of test item concentrations during the test period, the mean measured test item concentrations were calculated as the time-weighted geometric means over all measurements, with the exception of the nominal test item concentration of 10 μg/L. The substance had significant effects on the growth of the tested green algal species, with the following effect levels on the growth rate: 72-hour ErC10 = 0.0027 mg/L; 72-hour ErC50 = 0.027 mg/L.

Description of key information

72-hour ErC10 = 0.0027 mg/L (Pseudokirchneriella subcapitata, OECD TG 201, GLP)

72-hour ErC50 = 0.027 mg/L (Pseudokirchneriella subcapitata, OECD TG 201, GLP)

Key value for chemical safety assessment

EC50 for freshwater algae:

0.027 mg/L

EC10 or NOEC for freshwater algae:

0.003 mg/L