Dipotassium methanedisulphonate

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:

04 September 2017 - 16 March 2018

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 (control, G1) and 100 mg/L (G2)
- Sampling method: A volume of 2.0 mL sample solution from the group G1 and G2 were transferred into separate volumetric flask of 10 mL capacity. A volume of 0.5 mL 1:1 nitric acid solution was added to each flask and the volume of each flask was made up to the mark with reconstituted water.
- Sample storage conditions before analysis: not applicable

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An amount of 100 mg dipotassium methanedisulphonate was transferred to a 10 mL volumetric flask and the volume was made up to the mark with the algal cultured medium to obtain the nominal concentration of 10.0 mg/mL (stock A). The volume of 1600 µL from stock A and 1.4 mL alga culture were transferred using a micropipette and diluted to 160 mL with sterile algal culture medium to obtain the test concentration of 100.0 mg/L. Only 1.4 mL alga culture was transferred using a micropipette and diluted to 160 mL with sterile algal culture medium for control group. Six replicates for the control group and the treatment groups were used. The experiment was conducted in 250 mL conical flasks.
- Controls: sterile algal culture medium (negative) and potassium dichromate (positive).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection, 10801, University of Boulevard, Manassas, Virginia, 20110-2209, USA
- Method of cultivation: static condition

ACCLIMATION
- Acclimation period: 48 h
- Culturing media and conditions (same as test or not): OECD medium was used. The pre-culture was incubated under the same conditions as those required for the test and were used when growing exponentially (5 x 10E+03 - 10E+04 cells/mL). The temperature for pre-culture ranged between 21-23ºC.
- Any deformed or abnormal cells observed: not reported.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None

Test temperature:

21-23ºC

pH:

7.84-8.02

Nominal and measured concentrations:

Nominal concentrations: 0.0 (control) and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile conical flask
- Material, size, headspace, fill volume: glass, 250 mL capacity, 160 mL fill volumen
- Aeration: The cells in the cultured flasks were maintained in suspension by agitating the test flasks continuously at 100 ± 2 rpm, using an orbital shaker during the test period.
- Initial cells density: 6234 cells/mL
- Control end cells density: 748750 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (OECD medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the OECD medium was prepared as per the OECD guideline 201.
- 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: 6470 – 6593 (± 15% of the mean value) obtained with an universal UV-free white fluorescent lamp.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell concentration of each replicate was determined once, at a regular interval of 24 h, by manual counting using a haemocytometer (Hausser Scientific, U.S.A.) and a binocular microscope (Nikon)
- Other:
Temperature: measured at 24 h intervals.
Light intensity: measured at 24 h intervals.
pH: measured at the beginning and at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: N/A
- Justification for using less concentrations than requested by guideline: Based on percent inhibition of biomass/growth rate results at the highest concentration, i.e. 100 mg test item/L, obtained in a preliminary range finding study.
- Range finding study
- Test concentrations: 0.0 (control), 0.01, 0.1, 1.0, 10.0 and 100.0 mg/L
- Results used to determine the conditions for the definitive study: The percent inhibition of biomass was 0.47, 0.35, 0.82, 1.18 and 1.65, and the percent inhibition of growth rate was 0.00, -0.15, 0.15, 0.15 and 0.15 at the test concentrations of 0.01, 0.1, 1.0, 10.0 and 100.0 mg/L, respectively.

Reference substance (positive control):

yes

Remarks:

(potassium dichromate)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

(Yield)

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: (biomass (cell growth), growth rate and yield)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: (biomass (cell growth), growth rate and yield)

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: none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72h-ErC50: 1.29 mg/L (95% CL: 1.12-1.49)
- 72h-EyC50: 0.47 mg/L (95% CL: 0.41-0.54)

Table 1: Mean Values of Algal Biomass, Yield and Specific Growth Rate

Group	Test Concentration (mg/L)	Mean Number of Cells Count/mL at					Specific Growth Rate (0 – 3)
		0 h	24 h	48 h	72 h	Yield
G1	0.0 (Control)	6234	33333	164583	748750	742516	1.60
G2	100.0		30000	158750	742083	735849	1.59

Table 2: Algal Cell Count, Yield and Specific Growth Rates

Group	Test Concentration (mg/L)	Replicate	Cell Count at			Yield	Specific Growth Rate
			24 h	48 h	72 h	Cells/mL	0-1	1-2	2-3	0-3
G1	0.0 (Control)	R1	30000	162500	742500	736266	1.57	1.69	1.52	1.59
		R2	32500	165000	747500	741266	1.65	1.62	1.51	1.60
		R3	25000	162500	722500	716266	1.39	1.87	1.49	1.58
		R4	40000	170000	747500	741266	1.86	1.45	1.48	1.60
		R5	37500	160000	770000	763766	1.79	1.45	1.57	1.61
		R6	35000	167500	762500	756266	1.73	1.57	1.52	1.60
G2	100.0	R1	35000	150000	775000	768766	1.73	1.46	1.64	1.61
		R2	32500	160000	700000	693766	1.65	1.59	1.48	1.57
		R3	27500	170000	725000	718766	1.48	1.82	1.45	1.59
		R4	37500	162500	750000	743766	1.79	1.47	1.53	1.60
		R5	22500	165000	767500	761266	1.28	1.99	1.54	1.60
		R6	25000	145000	735000	728766	1.39	1.76	1.62	1.59

Note: Values are total number of cells counted per mL

Yield = Biomass at the end of Exposure period (72 h) – Biomass at the start of the Exposure period

*Initial Mean Cell Count: 6234

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 < 35%)

Conclusions:

In the Pseudokirchneriella subpicata green algae toxicity test the 72h-ErC50 of the test substance was determined to be higher than 100.0 mg/L.

Executive summary:

An Algae Growth Inhibition Test was performed with Pseudokirchneriella subpicata 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 to be soluble in OECD medium so no vehicle was used for the study. A preliminary range finding study, with 2 replicates for test item and 3 replicates for control, was conducted with the test concentrations of 0.0 (control), 0.01, 0.1, 1.0, 10.0 and 100.0 mg/L, resulting in percent inhibitions of growth rate of 0.00, -0.15, 0.15, 0.15 and 0.15, respectively. Based on these results, the test concentrations selected for the main study were 0.0 (control) and 100.0 mg/L. Six replicates for the control group and the treatment group were used. 1.4 mL of algal culture was inoculated into the test vessel for each replicate to obtain an initial mean cell concentration of 6234 cells/mL. A validated analytical method based on ICP-OES was used to monitor the concentration and stability of the main active ingredients in the test solution at 0 and 72 h. The active ingredient concentration in the test media was within acceptable limit (> 80% of nominal concentration). All validity criteria were fulfilled. There was no significant alteration observed in algal growth (biomass), yield and growth rate of the test item exposed group as compared to control group. The test substance does not inhibit the growth of alga (Pseudokirchneriella subcapitata) up to the concentration of 100.0 mg/L. The ErC50, NOEC and LOEC value was greater than 100.0 mg/L.

Description of key information

Key study. Test method according to OECD 201, GLP study. In the Pseudokirchneriella subpicata green algae toxicity test the 72h-ErC50 of the test substance was determined to be greater than 100.0 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Key study. An Algae Growth Inhibition Test was performed withPseudokirchneriella subpicatagreen 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 to be soluble in OECD medium so no vehicle was used for the study. A preliminary range finding study, with 2 replicates for test item and 3 replicates for control, was conducted with the test concentrations of 0.0 (control), 0.01, 0.1, 1.0, 10.0 and 100.0 mg/L, resulting in percent inhibitions of growth rate of 0.00, -0.15, 0.15, 0.15 and 0.15, respectively. Based on these results, the test concentrations selected for the main study were 0.0 (control) and 100.0 mg/L. Six replicates for the control group and the treatment group were used. 1.4 mL of algal culture was inoculated into the test vessel for each replicate to obtain an initial mean cell concentration of 6234 cells/mL. A validated analytical method based on ICP-OES was used to monitor the concentration and stability of the main active ingredients in the test solution at 0 and 72 h. The active ingredient concentration in the test media was within acceptable limit (> 80% of nominal concentration). All validity criteria were fulfilled. There was no significant alteration observed in algal growth (biomass), yield and growth rate of the test item exposed group as compared to control group. The test substance does not inhibit the growth of alga (Pseudokirchneriella subcapitata) up to the concentration of 100.0 mg/L. The ErC50, NOEC and LOEC value was greater than 100.0 mg/L.