Violet sodium polysulfide aluminosilicate with a SOD-type framework structure

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:

13 April 2020 - 01 January 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD Series on Testing and Assessment no. 23

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.0 (control) and 100.0 (nominal) mg/L.
- Sampling method: samples of 10 mL were collected from all test concentrations in duplicates at 0 and 72 hours. Samples of 15 mL were collected from further replicates at 0, 24 and 72 hours.
- Sample storage conditions before analysis: all samples were analysed on the same day of collection from the respective test vessels.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution was prepared by weighing required quantity of test item in a beaker; to this, a small volume of OECD alga medium was added and stirred well using a glass rod; after complete solubility of test item, test formulation was transferred to measuring cylinder, beaker was rinsed with OECD alga medium and transferred again to measuring cylinder. Rinsing process was repeated until the complete transfer of test contents.
The required test concentrations were prepared by adding appropriate amount of stock solutions and made up to required volume using OECD alga medium. 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, no filtration or other separation methods were used (measurements account for both the soluble and non-soluble fractions).
- Evidence of undissolved material: at 24, 48, and 72 hours, fine particles were observed at the bottom of conical flask at the test concentration of 100.0 mg/L.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- 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 two flasks containing 100 mL of culture medium) during 3 days prior to each test. The algal cell density of pre-cultures was 45225 and 43350 cells/mL at the start of the dose range-finding and main tests, respectively. The biomass increased by a factor of 8.0 (day 2) and 21.1 (day 3) for the dose range-finding and by a factor of 8.1 (day 2) and 21.8 (day 3) for the main test.
- Any deformed or abnormal cells observed: no.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22.1 to 22.7°C

pH:

8.06 to 8.22 (0 h) and 8.15 to 8.31 (72 h)

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 100 mg/L.
Measured concentrations: Since measured values by ICP-MS were within ± 20 % of the initial values, nominal concentrations are reported. However, at tested concentration of 100 mg/L, the limit of saturation was found to be, 8.94 to 10.75 mg test item/L in filtered sample and 8.20 to 11.89 mg test item/L in the centrifuged sample, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: sterilised Erlenmeyer flasks with air permeable stoppers
- Material, size, headspace, fill volume: glass, 250 mL capacity, the minimum volume of the test solution at the initiation of the test in the test vessel was 100 mL and in order to shorten the light path the test volume was made to 25 mL in each flask.
- Initial cells density: 5660 cells/mL (average)
- Control end cells density: 946000 cells/mL (average)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM / WATER PARAMETERS
- Standard medium used: yes (OECD medium as per TG 201)
- Source/preparation of dilution water: sterilized deionized water
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: constant illumination
- Light intensity and quality: 8223 to 8928 Lux, white fluorescent lamps (irradiation was above 120 µE/m2sec as recommended per OECD GD 23 for coloured substances).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber; samples collected were loaded on a 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).
Cell Density (cells/mL) = Average cell count × 10000 (CCF) × Dilution factor
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- 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: Under test conditions, average inhibition growth rates were 1.89, 3.78, 5.09, 6.40, 8.58 and 9.88, respectively. No toxic effects were observed up to the highest concentration tested. Therefore a limit test was performed.

Reference substance (positive control):

yes

Remarks:

(3,5-dichlorophenol 97%)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

8.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: No effects were observed up to the limit of saturation of the test item in test media.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 8.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: No effects were observed up to the limit of saturation of the test item in test media.

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no.
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: during the main test, fine particles were observed at the bottom of the flasks after 24, 48 and 72h. On analytical verification at tested concentration of 100 mg/L, the limit of saturation was found to be, 8.94 to 10.75 mg test item/L in filtered sample and 8.20 to 11.89 mg test item/L in the centrifuged sample, respectively.
- Effect concentrations exceeding solubility of substance in test medium: yes.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50 (0-72h) = 1.07 mg/L (0.91 – 1.27)

Reported statistics and error estimates:

The 72 hours EC10, EC20 and EC50 values were not calculated for growth rate and yield as it was a limit test.

Table 1. Mean cell density after exposure during main study (limit test).

Group	Test Conc.  (mg/L)	Replicate	Mean Cell Density (104Cells/mL)
			Observation Periods
			24 hour	48 hour	72 hour
G1	0.0	1	3.58	29.15	92.13
		2	3.85	28.33	89.38
		3	3.85	28.05	91.30
		4	4.13	28.60	92.68
		5	4.13	27.78	92.13
		6	3.85	29.15	90.48
G2	100.0	1	3.85	25.03	88.00
		2	3.30	25.85	84.43
		3	3.03	25.58	85.53
		4	3.30	27.78	86.08
		5	3.58	26.95	85.25
		6	3.30	26.40	87.45

 

Table 2: Average specific growth rate during main study (limit test).

Group	Test Conc. (mg/L)	Replicate	Growth Rate				Mean Growth Rate
			Observation Intervals (Days)
			0 to 1	1 to 2	2 to 3	0 to 3
G1	0.0	1	1.84	2.10	1.15	1.70	1.69
		2	1.92	2.00	1.15	1.69
		3	1.92	1.99	1.18	1.69
		4	1.99	1.94	1.18	1.70
		5	1.99	1.91	1.20	1.70
		6	1.92	2.02	1.13	1.69
		Mean	1.93	1.99	1.16	1.69
		±SD	0.05	0.07	0.02	0.00
		CV (%)	2.78	3.39	2.11	0.27
G2	100.0	1	1.92	1.87	1.26	1.68	1.68
		2	1.76	2.06	1.18	1.67
		3	1.68	2.13	1.21	1.67
		4	1.76	2.13	1.13	1.67
		5	1.84	2.02	1.15	1.67
		6	1.76	2.08	1.20	1.68
		Mean	1.79	2.08	1.16	1.68
		±SD	0.05	0.06	0.03	0.00
		CV (%)	2.58	2.66	2.94	0.26

^{±SD: Standard deviation; CV: Coefficient of Variation.}

 

Table 3. Yield of alga during main study (limit test).

Group	Test Conc. (mg/L)	Replicate	Cell density (Cells/mL)		Yield (72 hours)
			0 hour	72 hour
G1	0.0	1	5660	921250.00	915590.50
		2	5660	893750.00	888090.50
		3	5660	913000.00	907340.50
		4	5660	926750.00	921090.50
		5	5660	921250.00	915590.50
		6	5660	904750.00	899090.50
			Mean	913458.33	907798.83
			±SD	12349.51	12349.51
G2	100.0	1	5660	880000.00	874340.50
		2	5660	844250.00	838590.50
		3	5660	855250.00	849590.50
		4	5660	860750.00	855090.50
		5	5660	852500.00	846840.50
		6	5660	874500.00	868840.50
			Mean	861208.33	855548.83
			±SD	13630.31	13630.31

^{±SD: Standard deviation.}

 

Table 4. Percent inhibition in yield, cell density and growth rate during main study (limit test)

Group	Test Conc. (mg/L)	% Inhibition in Yield	% Inhibition in Cell Density	% Inhibition in Growth Rate
G1	0	-	-	-
G2	100.0	5.76	5.72	1.13

 

Table 5. Acceptance criteria for control during main study (limit test).

Daily average growth rate	±SD	% CV out of the daily growth rates
1.70	0.49	28.89
1.69	0.47	27.72
1.69	0.45	26.36
1.70	0.45	26.73
1.70	0.43	25.54
1.69	0.49	28.79
Mean % of CV 0 – 72 hours		27.34
Mean % CV of specific growth rate 0-72 hours		0.27

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 0.27 during limit test (validity criterion: should not exceed 7%).

 

Table 6. Biomass increase during the whole test period

Initial biomass (cells/mL)	Cell density on day 3 (cells/mL)	Biomass increase Factor
			5660	893750	157.92
5660	913000	161.32
5660	926750	163.75
5660	921250	162.78
5660	904750	159.86
Mean	913458.33	161.40

^{±SD: Standard deviation.}

Table 7. Summary of test concentration analysis during the 72-hour alga growth inhibition test

Group	0 hour Fresh Solution			72 hour Spent Solution
	Nominal Concentration (mg/L)	Conc. Obtained                         (mg/L)	(%) Recovery	Nominal Concentration (mg/L)	Conc. Obtained                          (mg/L)	(%) Recovery
G1	0.0	-	-	0.0	-	-
G2	100.0	103.43	103.43	100.0	108.20	108.20

 

Table 8. Temperature and light intensity during pre-culture and exposure - main study

	Temperature of the surrogate (°C)	Light intensity (Lux)				Revolutions per minute (rpm)
During Acclimatization (pre-culture and Exposure period)
0	22.7	8854	8571	8453	8765	100
1	22.3	8733	8617	8758	8935	100
2	22.5	8671	8529	8750	8771	100
3	22.2	8598	8724	8751	8834	100
4	22.7	8115	8217	8134	8226	100
5	22.6	8211	8314	8111	8139	100
6	22.3	8631	8237	8156	8227	100
Mean	22.5	8545	8458	8445	8557	-
±SD	0.2	274.9	200.6	310.0	342.0	-
Min	22.2	8111
Max	22.7	8935

^{-: Not applicable; SD: Standard deviation; Min: Minimum; Max: Maximum.}

 

Table 9. pH of test medium during the 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	0.0	8.15	8.18	8.22	8.2	8.19	8.22	8.28	8.25	8.31	8.28	8.24	8.27
G2	100.0	8.09	8.06	8.10	8.11	8.08	8.11	8.19	8.16	8.23	8.17	8.21	8.15
	Min	8.06						8.15
	Max	8.22						8.31

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 72h-ErC50 and NOEC of the test item based on growth rate were found to be above the saturation limit of the test item.

Executive summary:

An Algae Growth Inhibition Test was performed with Pseudokirchneriella subcapitata green algae on the test item, over a period of 72 h in static conditions, according to OECD TG 201, under GLP conditions. 

Based on an in-house solubility test, the test item was found to be soluble up to 100 mg/L. Based on the performed preliminary range-finding test performed at 0 (control), 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L, where no toxic effects were observed at any dose tested, a Limit Test was carried out using only one test concentration of 100 mg/L (nominal) and one control group. OECD medium was used for algal culturing. The test design included six replicates at test concentration and six replicates for the untreated control. Test medium alone and 3,5-dichlorophenol 97% were used as negative and positive controls, respectively. 

A validated analytical method based on ICP-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 percent of the nominal concentrations. However, during the main test, fine particles were observed at the bottom of the conical flask at the test concentration of 100.0 mg/L after 24, 48 and 72 hours. Therefore, additional replicates were prepared for analytical monitoring. On analytical verification at the concentration of 100 mg/L, the limit of saturation was found to be, 8.94 to 10.75 mg test item/L in filtered sample and 8.20 to 11.89 mg test item/L in the centrifuged sample, respectively.

Under test conditions and based on nominal concentrations, the 72-hour No Observed Effect Concentration (NOEC) was determined to be 100.0 mg/L, and the Lowest Observed Effect Concentration (LOEC) was >100.0 mg/L. The 72 hours EC50 value for growth rate (ErC50) and yield (EyC50) were determined to be >100.0 mg/L. All validity criteria were met.

Therefore, no effects were observed up to the limit of saturation of the test item in test media.

Description of key information

Key study: Method according to OECD 201, GLP study. Under test conditions and based on nominal concentrations, the 72-hour No Observed Effect Concentration (NOEC) was determined to be 100.0 mg/L, and the Lowest Observed Effect Concentration (LOEC) was >100.0 mg/L. The 72 hours EC50 value for growth rate (ErC50) and yield (EyC50) were determined to be >100.0 mg/L. All validity criteria were met. Based on analytical verification, no effects were observed up to the limit of saturation (8.94 to 10.75 mg test item/L) of the test item in test media.

Key value for chemical safety assessment

EC50 for freshwater algae:

8.94 mg/L

EC10 or NOEC for freshwater algae:

8.94 mg/L