Bis (3‐(diethylamino)‐7‐hydroxy‐5‐phenylphenazinium) sulphate

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:

02 November 2019 – 03 March 2020

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.0 (control), 0.5, 1.3, 3.2, 8.0, 20.0 and 50.0 mg/L
- Sampling method: samples (plus retain samples) were taken from a randomly chosen replicate per treatment group at 0 hour and 72 hours in order to determine the concentration of test item during the test. A volume of 10 mL of sample solutions were collected from all test concentration in duplicates and diluted in the corresponding quantity of methanol before being analyzed.
- Sample storage conditions before analysis: All samples were analyzed on the same day of collection from the respective test vessels.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution was freshly prepared prior to exposure, by weighing 100 mg of test item in a beaker and diluted in 100 mL of OECD alga medium. After complete soluble of test item by mixing well using a glass rod, the test item formulation was transferred into a measuring cylinder and the beaker was rinsed with OECD alga medium and transferred again to the measuring cylinder. Rinsing process was repeated until the complete transfer of test contents. A homogeneous distribution of the test item within the stock solution was achieved by stirring using magnetic stirrer shortly before preparation of the test vessels. The required test concentrations were prepared by adding appropriate amount of stock solutions and made up to required volume using OECD alga medium.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga Pseudokirchneriella subcapitata
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection.

ACCLIMATION
- Acclimation period: 3 days (pre-culture).
- Culturing media and conditions (same as test or not): Inoculum was pre-cultured (in a flask containing 100 mL of culture medium) during 3 days prior to the test. The biomass increased by factor of 9.0 (on day 2) and 21.2 (on day 3) for the dose range finding study and by factor of 9.6 (on day 2) and 22.0 (on day 3) for the main study.
- Any deformed or abnormal cells observed: Not reported.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test temperature:

22.2 to 22.6ºC

pH:

7.99 to 8.18 (0 h) and 8.00 to 8.12 (72 h)

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 0.5, 1.3, 3.2, 8.0, 20.0 and 50.0 mg/L.
Measured concentrations for 0, 0.5, 1.3, 3.2, 8.0, 20.0 and 50.0 mg/L (initial time, T0h): -, 0.57, 1.42, 3.52, 9.03, 21.66 and 52.36 mg/L.
Measured concentrations for 0, 0.5, 1.3, 3.2, 8.0, 20.0 and 50.0 mg/L (final time, T72h): -, 0.55, 1.31, 3.50, 8.28, 20.37 and 51.42 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: erlenmeyer flask
- Material, size, headspace, fill volume: glass, 250 mL capacity, 100 mL fill volume.
- Aeration: Test vessels were covered by air permeable stoppers.
- Initial cells density: 5528 cells/mL
- Control end cells density: 933167 cells/mL (average)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes (OECD medium as per OECD TG 201)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the OECD medium was prepared as per the OECD TG 201.
- Culture medium different from test medium: No
- Intervals of water quality measurement: The temperature (culture medium) and light intensity (parallel to the level of culture medium was recorded at four random locations using a Lux meter) was monitored daily during pre-culture and exposure. The pH of the test concentrations was measured at the beginning (0 hours) and at the end (72 hours) of the exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Constant illumination
- Light intensity and quality: 8185 to 8923 lux with white fluorescent lamp. The maximum value recorded was slightly outside of the recommended range stated in the OECD TG 201 (4440-8880 lux). LIght intensity applied during test was near higher value of the menioned range because the test item is a coloured substance which can absorb light and limit growth of alga and then it could result in growth inhibition which would have been difficult to distighish from inherent toxicity.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: Counting chamber: Samples collected were loaded on haemocytometer and the cells were counted manually using microscope.
Cell density is obtained by calculating the average cell count [average cell counts calculated by summating the number of cells in all the four large corner squares (squares 1 to 4) and dividing the sum by 4] and multiplied with chamber conversion factor (CCF). Then the following formula was used for calculating cell density in mL:
Cell Density (cells/mL)= Average cell count × 10000 (CCF) × Dilution factor

TEST CONCENTRATIONS
- Spacing factor for test concentrations: geometric factor of 2.5
- Justification for using less concentrations than requested by guideline: Not applicable; 6 concentrations used for the main test.

- Range finding study
- Test concentrations: 0 (control), 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L.
- Results used to determine the conditions for the definitive study: At the tested concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L, the average inhibition growth rate was calculated to be 0.13, 1.24, 5.15, 20.75, 100 and 100% respectively.

Reference substance (positive control):

yes

Remarks:

(3,5-dichlorophenol 97% )

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

27.96 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval = [19.16 – 40.78]

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

8.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval = [6.47 – 10.27]

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.28 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval = [3.23 – 5.67]

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval = [4.08 – 7.69]

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.26 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval = [1.53 – 3.34]

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.41 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval = [0.86 – 2.30]

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Determined by SPSS statistical procedure for multiple sample comparison using individual replicate values of the specific growth rate.

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Determined by SPSS statistical procedure for multiple sample comparison using individual replicate values of the specific growth rate.

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Not reported.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not reported.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50 (0-72h) = 1.12 mg/L (0.95 – 1.33)

Reported statistics and error estimates:

The 72 hours EC10, EC20 and EC50 values were calculated for growth rate and yield using the Probit Finneys method. Probit regression line was drawn for assessed parameters by plotting the Probit inhibition values against the corresponding log concentration. Student’s t-test was performed for response variable to determine NOEC and LOEC.

Table 1: Mean cell density after exposure during main study

Group	Test Conc.  (mg/L)	R	Mean Cell Density (10^4Cells/mL)
			Observation Periods
			24 h	48 h	72 h
G1	0.0	R1	4.95	28.60	92.95
		R2	5.50	28.05	95.70
		R3	4.95	28.60	91.85
		R4	5.50	28.05	92.95
		R5	5.23	28.88	94.60
		R6	5.23	29.70	91.85
G2	0.5	R1	4.95	30.53	92.95
		R2	5.23	29.15	93.23
		R3	4.95	30.80	91.58
G3	1.3	R1	4.40	23.65	78.65
		R2	4.68	24.75	78.38
		R3	4.68	25.03	78.65
G4	3.2	R1	3.85	18.98	66.55
		R2	4.68	19.53	67.65
		R3	4.40	20.35	67.93
G5	8.0	R1	2.75	15.68	43.45
		R2	2.20	17.05	44.83
		R3	2.48	16.50	44.28
G6	20.0	R1	1.38	11.55	9.35
		R2	1.65	11.00	9.63
		R3	1.38	10.73	9.08
G7	50.0	R1	0.00	0.00	0.00
		R2	0.00	0.00	0.00
		R3	0.00	0.00	0.00

Table 2: Average specific growth rate during main study

Group	Test Conc. (mg/L)	R	Growth Rate				Mean Growth Rate
			Observation Intervals (Days)
			0 to 1	1 to 2	2 to 3	0 to 3
G1	0	1	2.19	1.75	1.18	1.71	1.71
		2	2.30	1.63	1.23	1.72
		3	2.19	1.75	1.17	1.70
		4	2.30	1.63	1.20	1.71
		5	2.25	1.71	1.19	1.71
		6	2.25	1.74	1.13	1.70
		Mean	2.25	1.70	1.18	1.71
		±SD	0.05	0.06	0.03	0.01
		CV	2.10	3.46	2.77	0.32
G2	0.5	1	2.19	1.82	1.11	1.71	1.71
		2	2.25	1.72	1.16	1.71
		3	2.19	1.83	1.09	1.70
		Mean	2.21	1.79	1.12	1.71
		±SD	0.03	0.06	0.04	0.00
		CV	1.41	3.39	3.32	0.19
G3	1.3	1	2.07	1.68	1.20	1.65	*1.65
		2	2.14	1.67	1.15	1.65
		3	2.14	1.68	1.15	1.65
		Mean	2.11	1.68	1.17	1.65
		±SD	0.03	0.01	0.03	0.00
		CV	1.65	0.47	2.63	0.04
G4	3.2	1	1.94	1.60	1.25	1.60	*1.60
		2	2.14	1.43	1.24	1.60
		3	2.07	1.53	1.21	1.60
		Mean	2.05	1.52	1.23	1.60
		±SD	0.10	0.08	0.03	0.00
		CV	4.85	5.50	2.09	0.23
G5	8.0	1	1.60	1.74	1.02	1.45	*1.46
		2	1.38	2.05	0.97	1.47
		3	1.50	1.90	0.99	1.46
		Mean	1.49	1.90	0.99	1.46
		±SD	0.11	0.15	0.03	0.01
		CV	7.47	8.11	2.69	0.36
G6	20.0	1	0.91	2.13	-0.21	0.94	*0.94
		2	1.09	1.90	-0.13	0.95
		3	0.91	2.05	-0.17	0.93
		Mean	0.97	2.03	-0.17	0.94
		±SD	0.11	0.12	0.04	0.01
		CV	10.83	5.82	-22.87	1.04
G7	50.0	1	0.0	0.0	0.0	0.0	0.0
		2	0.0	0.0	0.0	0.0
		3	0.0	0.0	0.0	0.0
		Mean	0.0	0.0	0.0	0.0
		±SD	0.0	0.0	0.0	0.0
		CV	0.0	0.0	0.0	0.0

±SD: Standard deviation; CV: Coefficient of Variation;*: Statistical Significance of >0.005.

Table 3: Yield of alga during main study

Group	Test Conc. (mg/L)	R	Cell density (Cells/mL)		Yield (72 hours)
			0 h	72 h
G1	0.0	1	5528	929500	923973
		2	5528	957000	951473
		3	5528	918500	912973
		4	5528	929500	923973
		5	5528	946000	940473
		6	5528	918500	912973
			Mean	933167	927639
			±SD	15426	15426
G2	0.5	1	5528	929500	923973
		2	5528	932250	926723
		3	5528	915750	910223
			Mean	925833	920306
			±SD	8840	8840
G3	1.3	1	5528	786500	780973
		2	5528	783750	778223
		3	5528	786500	780973
			Mean	785583	780056
			±SD	1588	1588
G4	3.2	1	5528	665500	659973
		2	5528	676500	670973
		3	5528	679250	673723
			Mean	673750	668223
			±SD	7276	7276
G5	8.0	1	5528	434500	428973
		2	5528	448250	442723
		3	5528	442750	437223
			Mean	441833	436306
			±SD	6921	6921
G6	20.0	1	5528	93500	87973
		2	5528	96250	90723
		3	5528	90750	85223
			Mean	93500	87973
			±SD	2750	2750
G7	50.0	1	5528	0	-5528
		2	5528	0	-5528
		3	5528	0	-5528
			Mean	0	0
			±SD	0	0

±SD: Standard deviation

Table 4: Percent inhibition in yield, cell density and growth rate during main study

Group	Test Conc. (mg/L)	% inhibition in Yield	% inhibition in Cell Density	% inhibition in Growth Rate
G1	0	-	-	-
G2	0.5	0.79	0.79	0.15
G3	1.3	15.91	15.82	3.36
G4	3.2	27.97	27.80	6.35
G5	8.0	52.97	52.65	14.58
G6	20.0	90.52	89.98	44.86
G7	50.0	100.00	100.00	100.00

Table 5: Acceptance criteria for control during main study

0 – 72 Hours Mean Coefficient of Variation of Section-By-Section Average Specific Growth Rate

Daily average growth rate	±SD	% CV out of the daily growth rates
1.71	0.51	29.75
1.72	0.54	31.47
1.70	0.51	30.19
1.71	0.55	32.43
1.71	0.53	30.91
1.70	0.56	32.82
Mean % of CV 0 – 72 hours		31.26

Biomass increase during the whole test period

Initial biomass (cells/mL)	Cell density on day 3 (cells/mL)	Biomass increase Factor
5528	929500	168.16
5528	957000	173.13
5528	918500	166.17
5528	929500	168.16
5528	946000	171.14
5528	918500	166.17
Mean	933166.67	168.82

±SD: Standard deviation

Table 6: Summary of test concentration analysis during the 72 hour alga growth inhibition test during main study with violet LCC6D dried

	0 Hour New Solution			72 hour Aged Solution
Group	Nominal concentration (mg/L)	Conc. obtained                          (mg/L)	Recovery (%)	Nominal concentration (mg/L)	Conc. obtained                          (mg/L)	Recovery (%)
G1	0.0	-	-	0.0	-	-
G2	0.5	0.57	113.00	0.5	0.55	109.00
G3	1.3	1.42	108.85	1.3	1.31	100.39
G4	3.2	3.52	109.85	3.2	3.50	109.22
G5	8.0	9.03	112.82	8.0	8.28	103.51
G6	20.0	21.66	108.30	20.0	20.37	101.83
G7	50.0	52.36	104.71	50.0	51.42	102.83

Table 7: Temperature and light intensity during pre-culture and exposure - main study

Day	Temperature of the surrogate (°C)	Light intensity (Lux)				Revolutions per minute (rpm)
		1	2	3	4
During Acclimatisation (pre-culture) & Exposure
0	22.5	8215	8326	8254	8357	100
1	22.4	8354	8337	8257	8421	100
2	22.3	8562	8389	8454	8356	100
3	22.3	8457	8185	8256	8347	100
4	22.5	8554	8621	8529	8923	100
5	22.6	8726	8524	8611	8715	100
6	22.2	8531	8429	8357	8562	100
Mean	22.4	8486	8402	8388	8526	-
±SD	0.1	164.2	141.9	145.8	221.8	-
Min	22.2	8185
Max	22.6	8923

Table 8: pH of test medium during main study

Group	Test Conc. (mg/L)	pH
		0 hours						72 hours
		R1	R2	R3	R4	R5	R6	R1	R2	R3	R4	R5	R6
G1	0	8.11	8.09	8.13	7.99	8.03	8.05	8.10	8.08	8.12	8.00	8.02	8.01
G2	0.5	8.12	8.11	8.13	-	-	-	8.11	8.10	8.12	-	-	-
G3	1.3	8.07	8.12	8.09	-	-	-	8.05	8.10	8.06	-	-	-
G4	3.2	8.12	8.18	8.14	-	-	-	8.08	8.12	8.11	-	-	-
G5	8.0	8.13	8.11	8.09	-	-	-	8.10	8.09	8.06	-	-	-
G6	20.0	8.07	8.06	8.03	-	-	-	8.03	8.02	8.01	-	-	-
G7	50.0	8.05	8.01	8.01	-	-	-	8.04	8.00	8.03	-	-	-
	Min	7.99						8.00
	Max	8.18						8.12

Validity criteria fulfilled:

yes

Remarks:

(increase of biomass in control during 72h>16 fold; coefficient of variation of the mean specific growth rate among replicates in control (t0-t72)<7%; the mean of the replicate coefficients of variation in section-by-section growth rate in control < 35%)

Conclusions:

In an Alga Growth Inhibition Test with Pseudokirchneriella subcapitata green algae, the growth rate inhibition effect concentration (72 h-ErC50) of the test substance was found to be 27.96 mg/L.

Executive summary:

An Algae Growth Inhibition Test was performed with Pseudokirchneriella subcapitata green algae on the test substance over a period of 72 h in static conditions according to OECD Guideline 201, following GLP. OECD medium was used for algal culturing. In the previous solubility study the test item was found soluble in OECD medium and thus no vehicle was needed. In a preliminary range finding study the average growth inhibition rate were 0.13, 1.24, 5.15, 20.75, 100 and 100% at the tested concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L, respectively. Based on these results, the definitive test was conducted with nominal test item concentrations of 0 (control), 0.5, 1.3, 3.2, 8.0, 20.0 and 50.0 mg/L (factor of 2.5). Test medium alone and 3,5-dichlorophenol 97% were used as negative and positive controls respectively. Three replicates were performed for each concentration of the test item, along with six replicates for the negative control group. A validated analytical method based on LC-MS/MS was used to monitor the concentration and stability of the active ingredient in the test solution. The test item was considered as stable in the testing conditions after 72 hours of exposure as the measured concentrations in samples were maintained within ± 20 per cent of the nominal concentrations. All validity criteria were fulfilled. The percent inhibitions in average specific growth rate were 0.15, 3.36, 6.35, 14.58, 44.86 and 100.0% and the percent inhibitions in yield were 0.79, 15.91, 27.97, 52.97, 90.52 and 100.0% at the tested concentrations of 0.5, 1.3, 3.2, 8.0, 20.0 and 50.0 mg/L respectively, when compared to the negative control. Based on these results, the growth rate inhibition effect concentrations were determined to be 72 h-ErC10 = 4.28 mg/L, 72 h-ErC20 = 8.15 mg/L and 72 h-ErC50 = 27.96 mg/L, and the yield inhibition effect concentrations were 72h-EyC10 = 1.41 mg/L, 72h-EyC20 = 2.26 mg/L and 72h-EyC50 = 5.60 mg/L. The 72 hours No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) were calculated to be 0.5 mg/L and 1.3 mg/L respectively.

Description of key information

Key study. Test method according to OECD 201, GLP study. The growth rate inhibition effect concentrations to Pseudokirchneriella subcapitata green algae were determined to be 72 h-ErC50 = 27.96 mg/L and 72 h-ErC10 = 4.28 mg/L. The NOEC and LOEC were calculated to be 0.5 mg/L and 1.3 mg/L respectively for the 72 h-study.

Key value for chemical safety assessment

EC50 for freshwater algae:

27.96 mg/L

EC10 or NOEC for freshwater algae:

4.28 mg/L