Pyrasulfotole

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 - 23 May 2003 (definitive study)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.4400 (Aquatic Plant Toxicity Test using Lemna spp. Tiers I & II))

Version / remarks:

1996 (draft version)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 4.69, 9.38, 18.75, 37.5, 75.0, and 150 μg/L (nominal concentrations)
- Sampling method: Samples of test item solutions, including controls, were taken on Day 0 (new solutions) and Day 7 (old solutions) to measure actual exposure concentrations. On Day 0 the test solutions were prepared as uniform batches. On this day a 260-mL aliquot of the uniform test solution was poured into each of the labeled test vessels. A 500 mL sample of the remaining, fresh prepared, test solution was collected for analysis at each concentration. A 20-mL sample of the stock solution was also submitted for analysis on Day 0. On Day 7, a portion of the old test solutions from all replicates within a test concentration was composited. A 500-mL sample of the composite was collected for measured concentration analysis at each test level.
- Sample storage conditions before analysis: no storage

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A primary stock solution (10 mg a.i./L) was prepared by dissolving 0.0108 g of the test item (95.4% a.i.) into 1 L of 20 x AAP media. This stock solution was used to prepare all treatment levels by spiking the appropriate amount of the stock solution into approximately 1.5 L of 20 x AAP contained in labeled 2 L volumetric flasks. Each flask was then brought to a volume of 2 L with 20 x AAP media.
- Controls: yes, medium control
- Chemical name of vehicle: no solvent was used

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: Duckweed
- Strain: Lemna gibba G3
- Source (laboratory, culture collection): Department of Horticultural Science at the University of Minnesota in St. Paul, Minnesota, USA
- Age of inoculum (at test initiation): 7-day old batch culture
- Method of cultivation: The culture was grown under test conditions in an environmental chamber at 25 ± 2.0 °C, with a 24-hour light photoperiod and a light intensity between 453 and 480 foot-candles (4.9 to 5.2 klux) provided by cool white fluorescent lamps

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

24.3 - 24.8 °C (mean 24.6 °C)

pH:

test start (day 0): 7.8 - 7.9
test end (day 7): 8.6 - 8.7

Conductivity:

1332 - 1342 μmhos/cm (control)
1330 - 1363 μmhos/cm (test concentration)

Nominal and measured concentrations:

control, 4.69, 9.38, 18.75, 37.5, 75.0, and 150 μg/L (nominal concentrations)
control, 4.14, 9.57, 18.6, 33.1, 74.2, and 152.5 μg ai./L (mean measured concentrations)

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes (environmental chamber)
Test vessel:
- Type (delete if not applicable): open (capped with sterile, petri dish lids)
- Material, size, headspace, fill volume: sterile, 650-ml borosilicate glass crystallization dishes (diameter of 125 mm and a height of 65 mm and depth approximately 25 mm), filled with 260 mL of test solution.
- Type of cover: petri dish lids
- Aeration: no aeration
- Agitation: no but the test vessels were randomized daily during the exposure period.
- Control end cells density: 110 fronds at test end (mean growth rate 0.01217; Mean calculated cumulative biomass 6456)
- No. of colonies per vessel: 13 fronds per vessel
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: destilled water
- Culture medium different from test medium: no
- Intervals of water quality measurement:The pH and conductivity were measured in all test solutions on Day 0 (new batch solutions) and on Day 7 (old solutions). A calibrated datalogger and thermocouple were used to monitor the test system temperature each hour. In addition, manual temperature readings were recorded daily using a calibrated thermometer.

OTHER TEST CONDITIONS
- Sterile test conditions: The study was conducted under axenic conditions. (medium was sterilized using a 0.20 µm filter)
- Adjustment of pH: The batch of nutrient media used to prepare the test solutions was adjusted to pH 7.5 with dilute hydrochloric acid
- Photoperiod: 24-hour light photoperiod
- Light intensity and quality: between 453 and 480 foot-candles (4.9 to 5.2 klux) provided by cool white fluorescent lamps

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The total number of fronds in each test vessel was manually counted on Days 0, 3, 5, and 7. Statistical analysis was performed for the endpoints of frond count (standing crop), growth rate, and cumulative biomass (area under the growth curve). At the same time, phytotoxicity observations were performed to determine the health of the plants in each test vessel.
- Determination of frond number: manual counting
- Determination of biomass: [dry weight] The plants from each replicate were dried at approximately 60 °C for at least 24 hours, transferred to a desiccator to cool to ambient temperature, and then weighed to determine the dry weight.


RANGE-FINDING STUDY
- Test concentrations: control, 0.001, 0.01, 0.1, 1.0 and 10.0 mg a.i./L (nominal)
- Results used to determine the conditions for the definitive study: Inhibition for frond counts and plant dry weights as compared to the control group were: 0.001 mg/L (-1, -3), 0.01 mg/L (-3, -14), 0.10 mg/L (80, 88), 1.0 mg/L (83, 88) and 10.0 mg/L (83, 88), respectively. The test item concentrations of the definitive test were based on these results.

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

110 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

9.57 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: growth rate and fronds count

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

18.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: growth rate and fronds count

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

42.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: cumulative biomass

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

18.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: cumulative biomass

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

33.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: cumulative biomass

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

44.9 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

30.2 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: fronds dry weight

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

9.57 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: fronds dry weight

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

18.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: fronds dry weight

Details on results:

- Decrease in frond size: A reduction in frond size was also noted in the treatment levels 33.1, 74.2, and 152.5 μg a.i./L on day 3. Observations on Day 5 revealed a reduction in frond size in the highest four treatment levels (18.6, 33.1, 74.2, and 152.5 μg a.i./L). On day 7 observations revealed reduced frond size in the highest four treatment groups
- Other: Observations made on Day 3 revealed a few occurrences of brown fronds in each of the highest three treatment levels (33.1, 74.2, and 152.5 μg a.i./L). Transparent fronds were seen in the 74.2 μg a.i./L treatment level. On day 5 brown fronds in the highest three treatment levels, and transparent fronds in the highest treatment level. On Day 7 brown and transparent fronds in the highest three treatment groups.
- Any stimulation of growth found in any treatment: yes, at 4.14 µg a.i./L (-2% inhibition)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no observations
- Others: Analytical and biological results in detail are summarized in tables 1-6 in section "any other information on results incl tables"

Results with reference substance (positive control):

no positive control was used

Reported statistics and error estimates:

Raw or transformed data from treatment groups were compared to the control group for normality and homogeneity of variance using the Shapiro-Wilks test and Levene's test of equal variance, respectively.If normality and homogeneity of variance were demonstrated for the raw or transformed values, then parametric analyses were conducted using analysis of variance (ANOVA) followed by Dunnett's test. If normality and/or homogeneity of variance were not demonstrated on raw or transformed values, nonparametric procedures were used. The ranks of the raw values were determined, and then an analysis of variance and a one-tailed Dunnett's test were performed on these ranks. Statistical analyses were conducted using PC-based computer programs (SAS version 8).
Further statistical analysis was conducted to determine if the inhibition on growth was significant. Statistical analysis was performed for the endpoints of frond count (standing crop), growth rate, and cumulative biomass (area under the growth curve). All treatment levels were compared to the control group for the above endpoints. Statistical analysis of the data did not passed the criteria for normality and homogeneity of variance for the standing crop and growth rate endpoints. Therefore, a nonparametric analysis was conducted for these endpoints. For the cumulative biomass endpoint, the data passed the criteria for normality and homogeneity of variance so a parametric analysis was conducted for this endpoint.

Mean measured calculations were based on the recoveries of the test solutions on days 0 and 7. This represents a range of 88 to 102% of the nominal test concentrations (Table 2). Recoveries for the control test solution were below the limit of quantitation (0.96 μg/L).

Percent inhibition of frond counts ranged from –2 to 69% as compared to the control. Percent inhibition for cumulative biomass and growth rate ranged from -1 to 75% and 0 to 55%, respectively.

Table 1: Measured Test Concentrations of test iteml During the Exposure of Lemna gibba G3

Measured Concentration (μg a.i./L)
Nominal Conc. (μg a.i./L)	Day 0 (new)	Day 7 (old)	Mean Standard Deviation a)	Percent of Nominal a)
Control	ND	ND	----	----
4.69	4.77	3.51	4.14 ± 0.89	88
9.38	10.3	8.83	9.57 ± 1.04	102
18.75	19.3	17.9	18.6 ± 0.99	99
37.5	35.4	30.8	33.1 ± 3.25	88
75.0	76.7	71.6	74.2 ± 3.61	99
150	157	148	152.5 ± 6.36	102
Lab Recovery b)
19.3	21.0	19.9	20.5 ± 0.85	106

ND = Not detected at or above the validated limit of quantitation (0.96 μg/L)

Conc. = Concentration

a) Calculations for mean, standard deviation, and percent of nominal concentration are based on

recoveries from Day 0 and Day 7

b) Percent recoveries for Day 0 and Day 7 were 109 and 103 %, respectively. Lab spike nominal concentration was 19.3 μg/L each day.

Table 2: Daily Test Temperature Range During the exposure of Lemna gibba G3 to test item

Temperature (°C)
Exposure Period (Hour)	Minimum	Maximum	Mean
1 – 24	24.3	24.6	24.5
25 - 48	24.5	24.6	24.5
49 – 72	24.4	24.6	24.5
73 – 96	24.5	24.8	24.6
97 – 120	24.5	24.7	24.6
121 – 144	24.5	24.6	24.6
145 – 168	24.6	24.7	24.6
Study	24.3	24.8	24.6

Daily temperature ranges reported are based upon the hourly temperatures recorded by the datalogger during the exposure period.

Table 3: pH and Conductivity Measurements During the Exposure of Lemna gibba G3 to test item

Mean Measured Concentration (μg a.i./L)	Day 0 (New)		Day 7 (Old)
	pH	Cond	pH	Cond
Control	7.9	1342	8.7	1332
4.14	7.9	1346	8.7	1330
9.57	7.9	1349	8.7	1341
18.6	7.9	1352	8.7	1348
33.1	7.9	1362	8.6	1350
74.2	7.9	1363	8.6	1357
153	7.8	1362	8.6	1359

Cond = Conductivity in μmhos/cm

Conductivity range = 1330 to 1363 μmhos/cm

Mean conductivity = 1350 μmhos/cm

pH range = 7.8 to 8.7

Table 4: Day 0 and 7 Frond Number During the Exposure of Lemna gibba G3 to test item

Mean Measured Concentration (μg a.i./L)	Replicate	Day 0	Day 7	Day 7 Mean	Standard Deviation	Percent* Inhibition
Control	A	13	108	110	3
	B	13	113
	C	13	109
4.14	A	13	136	113	26	-2
	B	13	117
	C	13	85
9.57	A	13	105	97	9	12
	B	13	88
	C	13	99
18.6	A	13	75	71	4	35
	B	13	67
	C	13	71
33.1	A	13	51	49	3	55
	B	13	51
	C	13	45
74.2	A	13	44	46	4	58
	B	13	51
	C	13	44
153	A	13	33	34	1	69
	B	13	33
	C	13	35

* Percent inhibition = 100 - ((mean frond count per test level/mean frond count of control) x 100)

Table 5: Day 7 Frond Count, Cumulative Biomass, and Growth Rate During the Exposure of Lemna gibba G3 to test item

Mean Measured Concentration (μg a.i./L)	Frond Counts	Percent Inhibition1	Mean Calculated Cumulative Biomass2	Percent Inhibition1	Mean Growth Rate3	Percent Inhibition1
Control	110	-	6456	-	0.01271	-
4.14	113	-2	6520	-1	0.01274 0
9.57	97	12	5752	11	0.01197	6
18.6	71*	35	5040	22	0.01010*	21
33.1	49*	55	3036*	53	0.00789*	38
74.2	46*	58	2192*	66	0.00755*	41
153	34*	69	1628*	75	0.00566*	55

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

1 Percent inhibition = 100 - ((mean value per test level/mean of controls) x 100).

2 Cumulative biomass is equal to the area under the growth curve

3 Growth rate is calculated from the frond count data

Table 6: Day 7 Frond Dry Weights During the Exposure of Lemna gibba G3 to test item

Mean Measured Concentration (μg a.i./L)	Replicate	Day 7 Dry Weight (grams)	Day 7 Mean (grams)	Standard Deviation (grams)	Percent* Inhibition
Control	A	0.0088	0.0107	0.0017
	B	0.0119
	C	0.0114
4.14	A	0.0120	0.0105	0.0019	0
	B	0.0111
	C	0.0084
9.57	A	0.0098	0.0091	0.0008	13
	B	0.0082
	C	0.0093
18.6	A	0.0070	0.0066**	0.0004	37
	B	0.0062
	C	0.0066
33.1	A	0.0046	0.0041**	0.0006	61
	B	0.0034
	C	0.0042
74.2	A	0.0034	0.0032**	0.0003	70
	B	0.0033
	C 0.0029
153	A	0.0025	0.0024**	0.0005	77
	B	0.0019
	C	0.0028

* Percent inhibition = 100 - ((mean dry weight per test level/mean dry weight control) x 100).

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

Table 7: Validity criteria for OECD 221 (2006)

Criterion from the guideline	Outcome	Validity criterion fulfilled
The doubling time of frond number in the control must be less than 2.5 days (60 h), corresponding to approximately a seven-fold increase in seven days and an average specific growth rate of 0.275 d^-1.	The frond number in the controls increased by an average factor of 8.5 in seven days.	yes

 

Validity criteria fulfilled:

yes

Remarks:

See Table 7 in "Any other information on results incl. tables".

Description of key information

EC50 (7 d) = 110 µg a.i./L (mean measured, Lemna gibba, growth rate, OPPTS 850.4400)

NOEC (7 d) = 9.57 µg a.i./L (mean measured, Lemna gibba, growth rate, OPPTS 850.4400)

Key value for chemical safety assessment

EC50 for freshwater plants:

110 µg/L

EC10 or NOEC for freshwater plants:

9.57 µg/L

Additional information

One experimental study is available investigating the effects of the substance to aquatic plants (M-107686-01-1). The study was performed according to OPPTS 850.4400 (GLP) under static conditions using Lemna gibba as test organism.

The duckweed Lemna gibba G3 was exposed for 7 days to the nominal (mean measured) concentrations: control (<1.00), 4.69 (4.14), 9.38 (9.57), 18.75 (18.6), 37.5 (33.1), 75.0 (74.2) and 150 (152.5) µg a.i./L. Growth was determined by frond counts on days 0, 3, 5, and 7. Samples of the test item solutions, including controls, were taken on Day 0 (new solutions) and Day 7 (old solutions) to measure actual exposure concentrations.

The mean measured concentrations were 4.14, 9.57, 18.6, 33.1, 74.2, and 152.5 μg a.i./L for the 4.69, 9.38, 18.75, 37.5, 75.0, and 150 μg/L nominal concentrations, respectively. The measured concentrations were in the range of 88 to 102% of the nominal test concentrations. Observations revealed reduced frond size in the highest four treatment groups (18.6, 33.1, 74.2, and 152.5 µg a.i./L) as well as brown and transparent fronds in the highest three treatment groups (33.1, 74.2, and 152.5 µg a.i./L). The NOEC and LOEC in the 7-day exposure of Lemna gibba G3 to the test item were 9.57 and 18.6 µg a.i./L, respectively for the standing crop, growth rate and frond dry weight endpoints. Frond dry weight was the most sensitive endpoint. The NOEC and EC50 for this endpoint were 9.57 and 30.2 µg a.i./L, respectively. However, since growth rate is the preferred endpoint according to ECHA Guidance R.7b (ECHA, 2016) it was used as the key value (EC50 (7 d): 110 µg/L; NOEC (7 d): 9.57 µg/L). All endpoints were based on the mean measured concentrations.