Pyrasulfotole

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Apr - 25 Apr 2004

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:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

1996 (draft)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: FIFRA Guideline 123-2

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal: 2.56, 6.4, 16.0, 40.0, 100 and 100 mg/L (buffered)
- Sampling method: samples were taken daily

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: All of the test solutions were prepared as 1-L uniform batches. The highest test solution concentration (100 mg/L) was prepared before a 60% serial dilution was prepared to 40, 16, 6.4 and 2.56 mg/L concentrations. All test solutions were brought to volume with 1xAAP media.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no precipitate

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Source (laboratory, culture collection): in-house culture (SC-206), originally obtained from The University of Texas at Austin (Austin, Texas, USA).
- Age of inoculum (at test initiation): 96 hours (in log phase growth stage)
- Method of cultivation: The batch culture was grown under test conditions in an environmental chamber at 24 ± 2 °C, with 24 hour light photoperiod, and a light intensity of approximately 400 foot-candles (4.3 klux).

ACCLIMATION
- Acclimation period: < 96 hours
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

24.1 to 24.8 °C

pH:

5.3 to 10.0 for all test levels and 7.1 to 10.0 excluding the 107 mg a.i./L test solution (prepared without buffered media) during the exposure period.

Conductivity:

88 to 105 μmhos/cm for all solutions excluding those prepared in buffered media.
353 to 371 μmhos/cm for the control and 106 mg a.i./L solutions prepared in buffered media.

Nominal and measured concentrations:

nominal: 2.56, 6.4, 16.0, 40.0, 100 and 100 (buffered) mg/L
measured: 2.6, 6.4, 16.4, 41.4, 107 and 106 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250-ml sterile borosilicate glass culture flasks filled with approximately 100 mL of test solution and capped with sterile inverted glass beakers.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250-mL sterile borosilicate glass culture flasks capped with sterile inverted glass beakers.
- Initial cells density: nominal density of 10,000 cells/mL
- Control end cells density: 2,969,000 cells/mL (1,499,900 cells/mL for buffer control)
- No. of vessels per concentration (replicates): 3 (buffered, 3 (non-buffered)
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, 1 x AAP

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: distilled water
- Conductivity: 88 to 105 μmhos/cm for all solutions excluding those prepared in buffered media.
353 to 371 μmhos/cm for the control and 106 mg a.i./L solutions prepared in buffered media.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: The pH at the highest tes concentration was significantly reduced when added to 1xAAP media. To assess the effects of this pH shift on relative toxicity, an additional high test solution (100 mg/L buffered) was prepared using buffered water.
- Photoperiod: 24 hour light
- Light intensity and quality: 400 foot-candles (4.3 klux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Z1 Beckman Coulter® particle counter with hemocytometer used to measure cell density every 24 h..

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: There were two range finding tests.
1. The nominal test concentrations were control, 0.001, 0.01, 0.1, 1.0 and 10 mg/L.
2. To generate data that would be more predictive of an EC50 the second study included nominal test concentrations of control, 0.01, 0.5, 1.0, 50.0 and 100 mg/L.
- Results used to determine the conditions for the definitive study: Inhibition of cell density compared to the control was 14, 35, 35, 30 and 37%, respectively in the first study. Inhibition of cell density compared to the control was 26, 5, 5, 50 and 71%, respectively in the second study. Thus, the definitive study was carried out with higher test substance concentrations.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

6.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

29.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 26.2 - 35.5 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

other: cell density

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

11.6 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

other: cell density

Remarks on result:

other: 95% CI (11.1 - 12.1 mg/L)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks:

cumulative

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

12 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks:

cumulative

Remarks on result:

other: 95% CI (11.2 - 12.7 mg/L)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no physical abnormalities observed
- 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

Reported statistics and error estimates:

1) Shapiro-Wilks test for normality and Levene's test for homogeneity of variance;
2) ANOVA followed by the Dunnett's Test to determine the No Observed Effect Concentration (NOEC);
3) The Logistic Model or Bruce/Versteeg Cumulative Normal Model using nonlinear (weighted) regression analysis to estimate the EC50 and EC25

Table 1: Measured test concentrations of the test substance during the exposure of Pseudokirchneriella subcapitata

Nominal Concentration (mg/L)	Day 0 Measured Concentration (mg/L)	Day 4 Measured Concentration (mg/L)	Mean Measured Concentration^a (mg/L)	Standard Deviation	Percent (%) Nominal
Control	<0.19	<0.19	<0.19	-
Control^b	<0.19	<0.19	<0.19	-
2.56	2.40	2.79	2.6	0.28	101
6.40	6.05	6.74	6.4	0.49	100
16	15.9	16.9	16.4	0.71	103
40	39.8	43.0	41.4	2.26	104
100	103.0	111.0	107	5.66	107
100^b	104.0	107.0	106	2.12	106
Lab Spikes
9.64	9.64	-	-	-	100
9.64	-	9.96	-	-	103

^aCalculations of measured concentrations were performed using Microsoft 7 Excel 2000 with unrounded data. Manual calculations may vary slightly.

^bTest solutions prepared in buffered (reconstituted) water

Table 2: 96-hour cell density, cumulative biomass, and growth rate during the exposure of Pseudokirchneriella subcapitata to the test substance

Mean Measured Concentration (μg a.i./L)	Mean Density (cells/ml x 10^4)	Percent (%) Inhibition^a	Mean Calculated Cumulative Biomass^b	Percent (%) Inhibition^a	Mean Growth Rate^c	Percent (%) Inhibition^a
Control	296.9		7530.5		0.059302
Control^d	149.9	-	-	-	-	-
2.6	283.4	5	7142.2	5	0.058808	1
6.4	239.1*	19	5761.7*	23	0.057044**	4
16.4	87.8*	70	2633.7*	65	0.046594*	21
41.4	6.3*	98	320.1*	96	0.019112*	68
107	2.4*	99	110.6*	99	0.008885*	85
106^d	9.1	-	-	-	-	-

* Statistically significant from control (Dunnett's one-tailed test; p ≤0.05)

** Statistically significant from control but not considered biologically significant

^a% Inhibition=100-((Treatment group parameter mean/control parameter mean)*100).

^bCumulative biomass is equal to the area under the growth curve.

^cGrowth rate is calculated from the cell density data.

^dTest solutions prepared in buffered (reconstituted) water excluded from statistical analysis.

Table 3: Validity criteria for OECD 201 (2006)

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	The biomass in the control cultures increased exponentially by a factor of 126.6 within a 72-hour test period.	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures was 8.75%	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests withPseudokirchneriella subcapitataandDesmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 7% in the test withPseudokirchneriella subcapitata.	yes

 

Validity criteria fulfilled:

yes

Remarks:

See Table 3 in "Any other information on results incl. tables".

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 Jun - 6 Jun 2003

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:

June 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

1996, draft

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: FIFRA Guideline 123-2

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from all test chambers on Day 0 (prior to the introduction of the test organisms to the test chambers) and on Day 4 (test termination).
- Sampling method: daily
- Sample storage conditions before analysis: analyzed same day as sampled

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: All of the test solutions were prepared as 1 L uniform batches. The high concentration test solution (100 mg/L) was prepared by adding 0.1049 g of the test substance to approximately 500 mL of enriched saltwater (ES) media in a 1 L volumetric flask. The flask was then brought to a volume of 1 L and sonicated for 50 minutes. The flask was then inverted several times to complete the mixing process. The remaining test solutions (40, 16, 6.4 and 2.56 mg/L) were prepared using a 60% serial dilution procedure from the high concentration. Each concentration was prepared by adding 400 ml of the next higher concentration to a 1-L volumetric flask containing approximately 500 mL of ES media. The solutions were then brought to volume (1 L) with ES media and inverted several times.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no precipitates

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Common name: saltwater diatom
- Source (laboratory, culture collection): culture was obtained from The Springborn Laboratories (Wareham, Massachusetts, USA) (SK-88)
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: The culture was maintained with the method of Stein (1973). Handbook of Phycological Methods: Culture Methods and Growth Measurements. Cambridge University Press., Cambridge, England.

ACCLIMATION
- Acclimation period: < 72 h
- Culturing media and conditions (same as test or not): ES media, same as test
- Any deformed or abnormal cells observed: no abnormalities observed

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

19.1 to 19.9 °C

pH:

7.4 to 8.8

Salinity:

26 to 28 ppt

Nominal and measured concentrations:

nominal: 2.56, 6.4, 16.0, 40.0 and 100 mg/L
measured: 2.53, 6.4, 16.4, 41.1 and 99.5 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL sterile borosilicate glass culture flasks and capped with sterile inverted glass beakers.
- Initial cells density: 10,000 cells/mL
- Control end cells density: 76,500,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, ES medium used

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: distilled water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes, adjusted to pH 8.0
- Photoperiod: 16 hours of illumination
- Light intensity and quality: 392 foot-candles (4.2 klux)
- Salinity (for marine algae): 26 to 28 ppt

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: light microscope and an Improved Neubauer hemocytometer to evalaute cell number after 24, 48, 72 and 96 h

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: control, 0.01, 0.1, 1.0, 10.0 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes, Inhibition of cell density compared to the controls was 5, -9, -13, 40 and 99%, respectively. Thus, concentrations between 2.56 mg/mL and 100 mg/mL were used.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

6.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

15.7 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 12.7 - 18.8 mg a.i./L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

6.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

16.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

9.2 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% CI 8.5 - 9.8 mg a.i./L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none observed
- Any stimulation of growth found in any treatment: no stimulation observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no precipitation observed

Reported statistics and error estimates:

1) Shapiro-Wilks test for normality and Levene's test for homogeneity of variance;
2) ANOVA followed by the Dunnett's Test to determine the No Observed Effect Concentration (NOEC);
3) The Logistic Model or Bruce/Versteeg Cumulative Normal Model using nonlinear (weighted) regression analysis to estimate the EC50 and EC25

For details on measured concentrations, cell densities, biomass and growth rates see attachment.

Mean measured calculations were based on the recoveries of the test solutions on Days 0 and 4. The mean measured concentrations were 2.4, 6.3, 16.3, 40.1, 104 and 102 mg a.i./L for the 2.56, 6.4, 16.0, 40.0, 100 and 100 (buffered) mg a.i./L nominal concentrations, respectively. This represents a range of 94 to 106% of the nominal test concentrations. Recoveries for the control test solutions were below the limit of quantitation (0.19 mg/L). No undissolved test substance was visually observed in the test vessels throughout the exposure period.

Table 1: Validity criteria for OECD 201 (2006)

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	The biomass in the control cultures increased exponentially by a factor of 43 within a 72-hour test period.	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures was 6.66%	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests withPseudokirchneriella subcapitataandDesmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 0.69% in the test withSkeletonema costatum.	yes

 

Validity criteria fulfilled:

yes

Remarks:

See Table 1 in "Any other information on results incl. tables".

Description of key information

Freshwater:

ErC50 (96 h) = 29.8 mg a.i./L (mean measured, Pseudokirchneriella subcapitata, OECD 201)

NOErC (96 h) = 6.4 mg a.i./L (mean measured, Pseudokirchneriella subcapitata, OECD 201)

Marine water:

ErC50 (96 h) = 15.7 mg a.i./L (mean measured, Skeletonema costatum, OECD 201)

NOErC (96 h) = 6.4 mg a.i./L (mean measured, Skeletonema costatum, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

29.8 mg/L

EC50 for marine water algae:

15.7 mg/L

EC10 or NOEC for freshwater algae:

6.4 mg/L

EC10 or NOEC for marine water algae:

6.4 mg/L

Additional information

All available studies were performed according to OECD 201 and GLP with analytical monitoring of the test item concentration.

For the key study (M-001513-01-1) the freshwater species Pseudokirchneriella subcapitata was exposed under static conditions (shaken cultures) for approximately 96 hours to the following nominal concentrations: Control, Buffered Control, 2.56, 6.4, 16.0, 40.0, 100 and Buffered 100.0 mg a.i./L. Mean measured concentrations were based on the recoveries of the test solutions on Days 0 and 4. The mean measured concentrations were 2.6, 6.4, 16.4, 41.4, 107 and 106 mg a.i./L. This represents a range of 100 to 107% of the nominal test concentrations. Based on regression calculations, cell density (standing crop) is the most sensitive endpoint to exposure to the test item. The 96-hour EC50 for cell density was 11.6 mg a.i./L. The 96-hour EC50 value for growth rate was 29.8 mg a.i./L and the NOEC for growth rate was 6.4 mg a.i./L.

The key study of the marine species Skeletonema costatum (M-122128-01-2) was exposed under static conditions (shaken cultures) for 96 hours to the following nominal concentrations: Control, 2.56, 6.4, 16.0, 40.0 and 100.0 mg a.i./L. Mean measured calculations were based on the recoveries of the test solutions on days 0 and 4. The mean measured concentrations were 2.53, 6.4, 16.4, 41.1 and 99.5 mg a.i./L. This represents a range of 99 to 103% of the nominal test concentrations. Based on regression calculations, cumulative biomass is the most sensitive endpoint to exposure to the test item. The 96-hour EC50 for cumulative biomass was 9.2 mg a.i./L. The 96-hour NOEC and LOEC were 6.4 mg a.i./L and 16.4 mg a.i./L, respectively for all endpoints. The 96-hour EC50 value for growth rate was 15.7 mg a.i./L and the NOEC for growth rate was 6.4 mg a.i./L.