Pyrasulfotole

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Mar - 21 Apr 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Version / remarks:

draft version 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: FIFRA 72-4 (b)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 3.13, 6.25, 12.5, 25.0 and 50.0 mg a.i./L (nominal)
- Sampling method: A 10 mL aliquot of test solution was sampled from composites of the control and each test level. Samples were taken on day 0, 5, 12, 19 and 21.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The 50 mg a.i./L test solution was prepared by adding 10 L of dilution water to a 22 L glass tank. The tank then received approximately 0.6 g of test item. An additional 2 liters of dilution water was then added to the tank and the contents were mixed for approximately 2 hours using an electric stirrer. The 25, 12.5, 6.25 and 3.13 mg/L nominal concentrations were prepared in 20 L tanks by adding 6 liters of the next higher concentration of test solution to a 12 L quantity of dilution water. The contents of each tank were manually mixed using a spatula prior to the preparation of the next, lower, test concentration.
- Controls:The control solution was prepared by adding 12 L of dilution water to a 20 L tank. No test item was added to the control tank.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No undissolved material was noted in any test concentration.
- Eluate: no
- Differential loading: yes

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: in-house culture maintained since August 6, 2003 originally Aquatic Biosystems, Fort Collins, CO (Lot No. ABS080603)
- Age of parental stock (mean and range, SD): 15-day old adults
- Feeding during test: yes
- Food type: Combination of green algae (Pseudokirchneriella subcapitata) and blended Tetrafin® flaked fish food
- Amount: The alga was dispensed equally to all vessels at a minimum rate of 2 x 10 E+08 algal cells/L of test solution.
- Frequency: fed once daily with algae and fed a slurry of Tetrafin® on Monday, Wednesday and Friday.
- Other: The water in the culture vessels was renewed three times per week. Daphnia culture techniques were based on those described by ASTM (1997). The culture area was maintained on a 16-hour daylight photoperiod, light intensity of approximately 40-70 foot-candle, and a temperature of 20 ± 2.0 °C. The water used to culture the Daphnia is from the same source as the water used in the test (referred to as dilution water).

ACCLIMATION
- not required (breeding under study conditions)

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: The Daphnia subculture for the test was initiated on March 16, 2004. The Daphnia in the subculture were held under the same conditions as the culture. Two neonates (<24 hours old) were impartially assigned to each often 400 mL beakers for a total of 20 neonates. The glass beakers were filled to a volume of 300 ml with culture water. First instar daphnids (<24 hours old) were used to begin the study on March 31, 2004. The daphnids used in the definitive study were from the third brood or older.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

164 - 172 mg/L as CaCO3 (control)
162 - 174 mg/L as CaCO3 (test concentrations)

Test temperature:

19.9 - 20.7 °C

pH:

8.1 - 8.5 (control)
8.1 - 8.5 (test concentrations)

Dissolved oxygen:

8.7 - 10.0 mg/L (control)
8.4 - 10.4 mg/L (test concentrations)

Conductivity:

408 - 540 µmhos/cm (control)
408 - 542 µmhos/cm (test concentrations)

Nominal and measured concentrations:

control, 3.13, 6.25, 12.5, 25.0 and 50.0 mg a.i./L (nominal)
<0.29, 3.20, 6.39, 12.8, 25.5 and 52.9 mg a.i./L (mean measured)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open
- Aeration: Dilution water was intensely aerated before use
- Material, size, headspace, fill volume: 1 L beakers containing 900 mL test solution (for sublethal effects and survivorship) and 250 mL beakers 200 mL test solution (for sublethal effects, survivorship, growth and reproduction)
- Renewal rate of test solution (frequency/flow rate): renewal of test solution: on every Monday, Wednesday and Friday of the exposure (with the exception of the day of the final day of the study)
- No. of organisms per vessel: 1 (for sublethal effects, survivorship, growth and reproduction) and 5 (for sublethal effects and survivorship)
- No. of vessels per concentration (replicates): 10 (for sublethal effects, survivorship, growth and reproduction) and 3 (for sublethal effects and survivorship)
- No. of vessels per control (replicates): 3 (for sublethal effects and survivorship) and 10 (for sublethal effects, survivorship, growth and reproduction)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dilution water consists of spring water blended with reverse osmosis water to produce hard (160 to 180 mg/L) water.
- Total organic carbon: 2.1 mg/L (spring water) and < 0.50 mg/L (reverse osmosis water -RO)
- Metals: Sodium: 120000 µg/L (spring) and 1400 µg/L (RO); Potassium: 1900 µg/L (Spring) and < 500 µg/L (RO); Magnesium: 9900 µg/L (spring) and <500 µg/L (RO); Calcium: 160000 µg/L (spring) and <1000 µg/L (RO); others: < 0.2 - < 100 µg/L (spring and RO);
- Pesticides: <0.10 µg/L - < 0.5 µg/L
- Chlorine: <0.003 mg/L
- Alkalinity: 111 - 152 mg/L (control); 107 - 153 mg/L (test concentrations)
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dissolved oxygen, pH, alkalinity, hardness and conductivity were measured in all levels on Days 0, on newly prepared solutions, and weekly thereafter on new and old test solutions. On the final day of the study (Day 21) analysis for these parameters was preformed on old solutions only. Temperature was recorded hourly (datalogger) and daily (manual).

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8-hours dark. A 30-minute transition period of lower lighting was provided at the initiation and termination of each light period to simulate dawn and dusk.
- Light intensity: ranged from 43 to 64 foot candles (mean = 563 lux) as measured by a Sper Scientific Model 840020 light meter.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobilization of Daphnia magna, Time of the first brood and man young per reproduction day for daphnia magna and the adult length and weight measured after 21 days

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
Range finding study
- Test concentrations: control, 0.08, 0.8, 8.0 and 80 mg/L
- Results used to determine the conditions for the definitive study: A significant effect on the number of adult daphnids immobilized occurred in the 80 mg/L concentration. No apparent effect occurred at all other test levels. The test item concentrations of the definitive test were based on the results of the range finding test.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

25.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

52.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

12.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

25.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

52.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Time to first brood

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 52.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Time to first brood

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

25.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Adult body weight

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

52.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Adult body length

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

52.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Adult dry weight

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 52.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Adult dry weight

Details on results:

- Observations on body length and weight: Growth endpoints included daphnid length and dry weight. A statistical significant reduction in length was detected for organisms in the 52.9 mg a.i./L test level when compared to the controls. This resulted in a NOEC and an LOEC of 25.5 and 52.9 mg a.i./L, respectively. For daphnid dry weight, no statistically significant difference for any test concentration was determined to exist when compared to the controls. Therefore, the NOEC was determined to be 52.9 mg a.i./L and the LOEC was determined to be > 52.9 mg a.i./L.
- Other biological observations: No sublethal effects were noted in the control, 3.20 and 12.8 mg a.i./L test concentrations. One organism was observed to be pale in the 6.39 mg a.i./L test concentration on study Days 4 and 5. By Day 6, the organism appeared normal and remained normal for the remainder of the test duration. Since no other sublethal effects were noted in this or the next higher test concentration (12.8 mg a.i./L), it was determined that this brief observation was not dose related. For the 25.5 mg a.i./L test concentration, one organism was noted to be pale and another quiescent. In the high test concentration (52.9 mg a.i./L) between 5 and 10 organisms were noted to be pale or quiescent. One 6.39 mg a.i./L organism were immobilized during the exposure period. Further, no organisms were noted to be immobile in the 3.20 and 12.5 mg a.i./L test concentrations. Four organisms were immobilized in the 25.5 mg a.i./L test concentration representing 16 percent of those organisms. At the 52.9 test level, 11 organisms were immobilized representing 44 percent of the test population at that concentration. The immobilization effects in the 25.5 and 52.9 mg a.i./L test concentrations were both statistically significant. No significant difference between the control and any test concentration was detected for the time to first brood, resulting in a NOEC of 52.9 mg a.i./L and a LOEC of > 52.9 mg a.i./L. The mean time to first brood ranged from 8.0 to 10.3 days for the control and all test levels. Statistical analysis of the young per adult reproduction day for Daphnia magna during the 21-day exposure indicated that only the 52.9 mg a.i./L test level had significantly reduced reproduction compared to the control. The resulting NOEC was 25.5 mg a.i./L with a LOEC of 52.9 mg a.i./L.
- Mortality of the control: One control organism were immobilized during the exposure period. This represents 4% of the organism being immobile which is well within the acceptability criteria of 10 percent (EPA 1986, OECD 1998)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
- Effect concentrations exceeding solubility of substance in test medium: no
- Other: Results on analytics and biological is summarized within the table 1 - table 4 in "Any other information on results incl. tables"

Results with reference substance (positive control):

no positive control was performed

Reported statistics and error estimates:

Statistical analyses were conducted using PC based computer programs (TOXSTAT) developed by West, Inc. and Gulley (1994) with conclusions of statistical significance based on a 95 percent confidence level (p ^ 0.05). Statistical tests were conducted: 1) chi-square test to test the normality of the data set; 2) Bartlett's test for homogeneity of variances. To determine if the treatment groups were significantly different from the control, the reproduction data, survival data and parent daphnid growth data (length and dry weight) were analyzed by a oneway analysis of variance (ANOVA) followed by the Dunnett's test, or a nonparametric analysis if the assumptions of normality and/or homogeneity of variance were not met.

Table 1: Measured Test Concentrations of test Item During the 21-day Exposure of Daphnia magna

Nominal Concentration (mg a.i./L)	Measured Concentration (mg a.i./L)
	DayO	Day 5	Day 12	Day 19	Day 21	Mean	Standard Deviation	Percent of Nominal
Control (old)	...	<0.29	<0.29	<0.29	<O.29	NA	NA	NA
Control (new)	<0.29	<0.29	O.29	<0.29	...
3.13 (old)	—	3.26	3.20	3.27	3.23	3.20	0.08	102
3.13 (new)	3.30	3.12	3.10	3.08	...
6.25 (old)	...	6.55	6.30	6.17	6.37	6.39	0.22	102
6.25 (new)	6.82	6.43	6.36	6.11	...
12.5 (old)	...	13.0	12.8	12.6	12.4	12.8	0.29	102
12.5 (new)	13.0	12.8	13.1	12.3	...
25 (old)	—	26.1	26.0	25.1	25.1	25.5	0.51	102
25 (new)	26.0	25.6	25.0	24.9	...
50 (old)	—	56.9	49.0	55.4	51.7	52.9	2.67	106
50 (new)	53.4	54.3	50.0	52.3	...

Limit of quantitation = 0.29 mg a.i./L

new = newly prepared batch solutions

old = solutions replaced during each renewal

Table 2: Day 21 Immobilization of Daphnia magna Exposed to Test Item

Mean Measured Concentration (mg a.i./L)	Replicate	Initial Number of Instars	Number of Adults on Day 21	Percent Survival (%)	Cumulative Immobility	Percent Immobilized (%)
Control	A	5	5	100	0	0
	B	5	5	100	0	0
	C	5	5	100	0	0
	D	1	0	0	1	100
	E	1	1	100	0	0
	F	1	1	100	0	0
	G	1	1	100	0	0
	H	1	1	100	0	0
	I	1	1	100	0	0
	J	1	1	100	0	0
	K	1	1	100	0	0
	L	1	1	100	0	0
	M	1	1	100	0	0
	Total	25	24	96	1	4
3.20	A	5	5	100	0	0
	B	5	5	100	0	0
	C	5	5	100	0	0
	D	1	1	100	0	0
	E	1	1	100	0	0
	F	1	1	100	0	0
	G	1	1	100	0	0
	H	1	1	100	0	0
	I	1	1	100	0	0
	J	1	1	100	0	0
	K	1	1	100	0	0
	L	1	1	100	0	0
	M	1	1	100	0	0
	Total	25	25	100	0	0
6.39	A	5	5	100	0	0
	B	5	5	100	0	0
	C	5	5	100	0	0
	D	1	1	100	0	0
	E	1	0	0	1	100
	F	1	1	100	0	0
	G	1	1	100	0	0
	H	1	1	100	0	0
	I	1	1	100	0	0
	J	1	1	100	0	0
	K	1	1	100	0	0
	L	1	1	100	0	0
	M	1	1	100	0	0
	Total	25	24	96	1	4
12.8	A	5	5	100	0	0
	B	5	5	100	0	0
	C	5	5	100	0	0
	D	1	1	100	0	0
	E	1	1	100	0	0
	F	1	1	100	0	0
	G	1	1	100	0	0
	H	1	1	100	0	0
	I	1	1	100	0	0
	J	1	1	100	0	0
	K	1	1	100	0	0
	L	1	1	100	0	0
	M	1	1	100	0	0
	Total	25	25	100	0	0
25.5	A	5	4	80	1	20
	B	5	4	80	1	20
	C	5	5	100	0	0
	D	1	0	0	1	100
	E	1	1	100	0	0
	F	1	1	100	0	0
	G	1	1	100	0	0
	H	1	1	100	0	0
	I	1	1	100	0	0
	J	1	1	100	0	0
	K	1	1	100	0	0
	L	1	1	100	0	0
	M	1	0	0	1	100
	Total	25	21	84*	4	16*
52.9	A	5	3	60	2	40
	B	5	4	80	1	20
	C	5	3	60	2	40
	D	1	0	0	1	100
	E	1	0	0	1	100
	F	1	1	100	0	0
	G	1	0	0	1	100
	H	1	0	0	1	100
	I	1	1	100	0	0
	J	1	0	0	1	100
	K	1	0	0	1	100
	L	1	1	100	0	0
	M	1	1	100	0	0
	Total	25	14	56*	11	44*

* Indicates the treatment group is significantly different from the control (p<0.05)

Table 3: Time to First Brood and Mean Young per Reproduction Day for Daphnia magna Exposed to Test Item

Mean Measured Concentration (mg a.i./L)	Replicate	Time to first Brood (days)	Total young	Number of Repro. Days	Young/Adult Repro. Day
Control	D	8	75	8	9.38
	E	8	214	14	15.29
	F	7	160	15	10.67
	G	7	182	15	12.13
	H	9	230	13	17.69
	I	9	165	13	12.69
	J	9	282	13	21.69
	K	11	203	11	18.45
	L	8	167	14	11.93
	M	8	190	14	13.57
	Mean	8.4	186.8	12.9	14.35
	SD	1.2	53.8	2.4	3.88
3.20	D	8	188	14	13.43
	E	10	223	12	18.58
	F	7	177	15	11.80
	G	7	175	15	11.67
	H	9	103	13	7.92
	I	7	218	15	14.53
	J	7	182	15	12.13
	K	9	195	13	15.00
	L	7	226	15	15.07
	M	9	219	13	16.85
	Mean	8.0	190.6	14.0	13.70
	SD	1.2	36.6	1.2	3.01
6.39	D	9	236	13	18.15
	E	9	196	13	15.08
	F	11	135	11	12.27
	G	8	220	14	15.71
	H	9	216	13	16.62
	I	11	187	11	17.00
	J	7	181	15	12.07
	K	11	216	11	19.64
	L	11	269	11	24.45
	M	9	202	13	15.54
	Mean	9.5	205.8	12.5	16.65
	SD	1.4	35.6	1.4	3.61
12.8	D	9	213	13	16.38
	E	9	191	13	14.69
	F	9	188	13	14.46
	G	7	193	15	12.87
	H	7	160	15	10.67
	I	9	240	13	18.46
	J	10	213	12	17.75
	K	8	223	14	15.93
	L	7	178	15	11.87
	M	9	203	13	15.62
	Mean	8.4	200.2	13.6	14.87
	SD	1.1	23.2	1.1	2.50
25.5	D		0	0	0.00
	E	8	231	14	16.50
	F	9	198	13	15.23
	G	11	138	11	12.55
	H	11	195	11	17.73
	I	9	193	13	14.85
	J	9	154	13	11.85
	K	7	167	15	11.13
	L	8	177	14	12.64
	M		0	0	0.00
	Mean	9.0	145.3	10.4	11.25
	SD	1.4	80.8	5.6	6.28
52.9	D	9	5	2	2.50
	E		0	0	0.00
	F	9	82	13	6.31
	G		0	0	0.00
	H	11	11	2	5.50
	I	11	110	11	10.00
	J	10	30	5	6.00
	K		0	0	0.00
	L	12	94	10	9.40
	M	10	116	12	9.67
	Mean	10.3	44.8	5.5	4.94
	SD	1.1	49.5	5.4	4.09

* Indicates the treatment group is significantly different from the control (p<0.05)

Table 4: Adult Length and Weight for Daphnia magna exposed to Test Item  

Mean measured Concentration (mg a.i./L)	Length (mm)		Weight (mg)
	Mean	Standard Deviation	Mean	Standard Deviation
Control	4.64	0.19	1.189	0.202
3.20	4.67	0.16	1.255	0.270
6.39	4.66	0.12	1.188	0.195
12.8	4.68	0.13	1.069	0.156
25.5	4.62	0.17	1.028	0.196
52.9	4.38*	0.24	1.007	0.201

*Indicates the treatment group is significantly different from the control (p<0.05)

Table 5: Validity criteria for OECD 211

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality of the parent animals in the control (femaleDaphnia) does not exceed 20% at the end of the test.	The mortality of the parent animals in the control (femaleDaphnia) was 96% at the end of the test.	yes
The mean number of living offspring produced per parent animalsurvivingat the end of the test is ≥ 60 in the control.	The mean number of living offspring produced per parent animalsurvivingat the end of the test was 186.8±3.9 in the control.	yes

 

Validity criteria fulfilled:

yes

Remarks:

See Table 5 in "Any other information on results incl. tables".

Description of key information

NOEC (21 d) = 12.8 mg a.i./L based on immobilisation (mean measured, Daphnia magna, OECD 211)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

12.8 mg/L

Additional information

One experimental study is available investigating the effects of the substance to the reproduction of aquatic invertebrates (M-249681-01-2). The study was performed according to OECD 211 (GLP) under semi-static conditions using Daphnia magna as test organism. The first instars of Daphnia magna (< 24 h old) under static renewal conditions were exposed to nominal (mean measured) concentrations of control (<0.29), 3.13 (3.20), 6.25 (6.39), 12.5 (12.8), 25.0 (25.5), and 50.0 (52.9) mg a.i./L (mg/kg) for 21 days. These measured concentrations represents a range 102 to 106 % of the nominal concentration. No undissolved test substance was observed in the test chambers.All measurements of the control treatment level were below the LOQ of 0.29 mg/L. All subsequent observations will refer to mean measured concentrations of the test solutions. One organism in the 6.39 mg a.i./L test concentration was observed to be pale for two days of the 21-day exposure. This observation was not considered to be a dose effect. For the 25.5 mg a.i./L test concentration, one organism was noted to be pale and another quiescent. In the high test concentration (52.9 mg a.i./L) between 5 and 10 organisms were noted to be pale or quiescent. The 21-day exposure to the test item resulted in a NOEC of 12.8 mg a.i./L and a LOEC of 25.5 mg a.i./L based on immobilization.