Diphenyl[4-(phenylsulfanyl)phenyl] sulfonium hexafluoroantimonate(1-)

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:

28 February - 12 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was conducted in accordance with International Guidelines (OECD 201) and in accordance with OECD Principles of Good Laboratory Practice as incorporated into the United Kingdom Statutory Instrument for GLP. All validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.010, 0.032, 0.10, 0.32 and 1.0 % v/v saturated solution
- Sampling method: Samples placed in amber bottles were taken from the control and each test group from the bulk test preparation at 0 hours, and from pooled replicates at 72 hours for quantitative analysis. Samples without algae were also taken at 24 and 48 hours from media run alongside the test.
- Sample storage conditions before analysis: All samples were taken for immediate analysis. Duplicate sets of samples were taken at each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 1100 mg test item added to 11 L of culture medium with stirring at 1500 rpm for 24 hours. After 24 hours, stirring was stopped and undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter; the first two liters passed through the filter were discarded. This gave a 100 % v/v saturated solution. A series of dilutions were made from this saturated solution to give the required test concentrations (0.010, 0.032, 0.1, 0.32, 1.0 and 10 % v/v saturated solutions). An aliquot (900 mL) of each of the 0.010, 0.032, 0.10, 0.32 and 1.0 % v/v saturated solution stock solutions were inoculated with algal suspension in order to give the required test concentrations.
- Controls: untreated control only

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, SAMS Research Services Ltd, Scottish Marine Institute, Oban, Scotland
- Age of inoculum (at test initiation): N/A
- Method of cultivation: Master cultures maintained in the laboratory by periodic replenishment of culture medium. Master cultures kept under constant agitation by orbital shaker (150 rpm) and contant illumination at 24 ± 1 °C.

ACCLIMATION
- Acclimation period: N/A
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: not reported, however, control algal cells were checked microscopically during the test and no abnormalities were found.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

N/A

Test temperature:

24 ± 1 °C

pH:

7.6 - 8.0

Dissolved oxygen:

N/A

Salinity:

N/A

Conductivity:

N/A

Nominal and measured concentrations:

Nominal: Control, 0.010, 0.032, 0.10, 0.32, 1.0% v/v saturated solution; Geometric mean measured concentrations: 0, 0.00052, 0.0017, 0.0031, 0.0082, 0.020 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks
- Type (delete if not applicable): plugged with polyuretherane foam bungs
- Material, size, headspace, fill volume: glass, filled with 100 mL test/control solution
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: 5.00 x 10E3 cells per mL
- Control end cells density: 1.08 x 10E6 cells per mL
- No. of organisms per vessel: 5.00 x 10E5
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): N/A

GROWTH MEDIUM
- Standard medium used: yes - AAP Medium
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionised water from Elga Optima 15+ or Elga Purelab Option R-15 BP
- Total organic carbon: N/A
- Particulate matter: N/A
- Metals: N/A
- Pesticides: N/A
- Chlorine: N/A
- Alkalinity: N/A
- Ca/mg ratio: N/A
- Conductivity: N/A
- Culture medium different from test medium: No
- Intervals of water quality measurement: 0, 72 h

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: culture medium only
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 7000 lux, warm white light
- Salinity (for marine algae): N/A

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Coulter Multisizer Particle Counter; three determinations per sample.
- Chlorophyll measurement: N/A
- Other: N/A

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study
- Test concentrations: First range finding study conducted at 0.10, 1.0, 10 and 100 % v/v saturated solution, all concentrations showed high levels of growth inhibition, so a second range finding test was conducted at 0.00010, 0.0010, 0.010, 0.10 and 1.0 % v/v saturated solution.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate, conducted under study number YJ31TQ

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.01 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: no confidence limits due to poor model fit

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.004 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.003 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

yield

Remarks on result:

other: 95% confidence limits: 0.0026 - 0.0030 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: no confidence limits due to poor model fit

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: no confidence limits due to poor model fit

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): yes
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: some healthy cells and cell debris observed at 0.0031 mg/L, very few healthy cells and few patches of cell debris observed at 0.0082 mg/L and no cells present at 0.020 mg/L
- 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
- EC50: ErC50: 1.5 mg/L (95% confidence limits: 1.3 - 1.7 mg/L)
- Other: The results from the positive control were within normal ranges for the reference item, based upon the last 5 years of in-house data (mean ErC50 of 1.4 mg/L).

Reported statistics and error estimates:

Statistical analysis of the growth rate and yield data was carried out for the control and all test concentrations using one way ANOVA incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control.

For each individual test vessel (mean values for yield), percentage inhibition was plotted against the logarithm test concentration and a line fitted by computerized interpolation using the Xlfit software package. ECx values were then determined from the equation for the fitted line.
Where appropriate 95% confidence limits for the EC50 values were calculated.
It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Table 1 Cell Densities and pH Values in the Definitive Test

Geometric Mean Measured Concentration (mg/L)	Replicate Flask	pH	Cell Densities (x10^4cells per mL)*			pH
		0 Hours	23 hours	53 hours	72 hours	72 hours
Control	R1	7.8	2.13	23.4	114	7.9
	R2		2.28	24.6	122
	R3		2.47	21.4	100
	R4		2.63	24.5	126
	R5		2.26	20.1	100
	R6		1.91	20.3	86.3
	Mean		2.28	22.4	108
0.00052	R1	7.7	2.58	22.8	116	7.9
	R2		2.56	25.6	129
	R3		2.75	24.5	128
	Mean		2.63	24.3	124
0.0017	R1	7.6	2.25	22.4	123	8.0
	R2		2.48	24.7	131
	R3		2.27	23.5	104
	Mean		2.33	23.5	119
0.0031	R1	7.6	1.67	8.67	31.3	7.9
	R2		1.71	9.62	36.7
	R3		1.94	9.42	38.5
	Mean		1.77	9.24	35.5
0.0082	R1	7.6	1.46	3.19	5.67	7.7
	R2		1.80	3.76	8.58
	R3		1.67	3.68	8.16
	Mean		1.64	3.54	7.47
0.020	R1	7.6	1.28	2.53	4.63	7.7
	R2		1.29	1.81	2.93
	R3		1.14	1.89	2.91
	Mean		1.24	2.08	3.49

* Cell densities represent the mean number of cells per mL calculated from the mean of 3 cell counts from each of the replicate flasks

Table 2 Daily Specific Growth Rates for the Control Cultures in the Definitive Test

 

Treatment		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 to 1	Day 1 to 2	Day 2 to 3
Control	R1	0.063	0.080	0.083
	R2	0.66	0.079	0.084
	R3	0.069	0.072	0.081
	R4	0.072	0.074	0.086
	R5	0.066	0.073	0.085
	R6	0.058	0.079	0.079
	Mean	0.066	0.076	0.083

Table 3 Inhibition of Growth Rate and Yield in the Definitive Test

 

Geometric Mean Measured Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (x10^4cells/mL)
		0 to 72 h	% Inhibition	0 to 72 h	Mean % Inhibition
Control	R1	0.075	-	114	-
	R2	0.076		122
	R3	0.074		99.6
	R4	0.077		125
	R5	0.074		99.7
	R6	0.072		85.8
	Mean	0.075		108
	SD	0.002		15.2
0.00052	R1	0.076	-1	116	-15
	R2	0.077	-3	128
	R3	0.077	-3	128
	Mean	0.077	-2	124
	SD	0.001	N/A	7.3
0.0017	R1	0.077	-3	123	-10
	R2	0.077	-3	130
	R3	0.074	1	104
	Mean	0.076	-2	119
	SD	0.002	N/A	13.7
0.0031	R1	0.057	24	30.8	67
	R2	0.060	20	36.2
	R3	0.060	20	38.0
	Mean	0.059	21	35.0
	SD	0.002	N/A	3.75
0.0082	R1	0.034	55	5.17	94
	R2	0.039	48	8.08
	R3	0.039	48	7.66
	Mean	0.037	50	6.97
	SD	0.003	N/A	1.57
0.020	R1	0.031	59	4.13	97
	R2	0.025	67	2.43
	R3	0.024	68	2.41
	Mean	0.027	65	2.99
	SD	0.004	N/A	0.989

SD: Standard Deviation

N/A: Not Applicable

A negative value for inhibition indicates an increase relative to the control

Validity criteria fulfilled:

yes

Remarks:

The control validity criteria are as follows: Cell concentration >16 times in 72 hours (actual: 216). Mean CV for section by section specific growth rate < 35% (actual: 12%). CV for average specific growth rate over the test period < 7% (actual: 3%).

Conclusions:

A study was performed according to OECD 201 (2006) to assess the toxicity of the test item to green algae (Pseudokirchneriella subcapitata). Algal cultures were exposed to aqueous solutions of the test item at nominal concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0% v/v saturated solution for 72 hours under static test conditions. Exposure of P. subcapitata to the test item gave the following results based on geometric mean measured test concentrations: 72-h ErC50 – 0.010 mg/L, 72-h ErC10 - 0.0019 mg/L.

Executive summary:

A study was performed according to OECD 201 (2006) to assess the toxicity of the test item to green algae (Pseudokirchneriella subcapitata). Due to light sensitive nature of the test item all media preparations were conducted under non-actinic light throughout the study. Following two range-finding tests, algal cultures were exposed to aqueous solutions of the test item at nominal concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0% v/v saturated solution for 72 hours under static test conditions, in constant illumination. Chemical analysis showed measured test concentrations to range from 0.000442-0.405 mg/L. The geometric mean measured test concentrations were determined to be 0.00052, 0.0017, 0.0031, 0.0082 and 0.020 mg/L. Exposure of P. subcapitatato the test item gave the following results based on geometric mean measured test concentrations: 72-h ErC₅₀– 0.010 mg/L, 72-h ErC₁₀ - 0.0019 mg/L. All validity criteria were met.

Description of key information

EC₅₀ - 0.010 mg/L, EC₁₀ - 0.0019 mg/L; OECD 201; Hull K (2019)

Key value for chemical safety assessment

EC50 for freshwater algae:

0.01 mg/L

EC10 or NOEC for freshwater algae:

0.002 mg/L

Additional information

A study was performed according to OECD 201 (2006) to assess the toxicity of the test item to green algae (Pseudokirchneriella subcapitata). Due to light sensitive nature of the test item all media preparations were conducted under non-actinic light throughout the study. Following two range-finding tests, algal cultures were exposed to aqueous solutions of the test item at nominal concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0% v/v saturated solution for 72 hours under static test conditions, in constant illumination. Chemical analysis showed measured test concentrations to range from 0.000442-0.405 mg/L. The geometric mean measured test concentrations were determined to be 0.00052, 0.0017, 0.0031, 0.0082 and 0.020 mg/L. Exposure ofP. subcapitatato the test item gave the following results based on geometric mean measured test concentrations: 72-h ErC₅₀– 0.010 mg/L, 72-h ErC₁₀- 0.0019 mg/L. All validity criteria were met.