Prometryn

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Oct 2000 to 31 Oct 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 87/302/EEC, L 133 Part C, pp.95-98

Version / remarks:

Edition 1988

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

PREPARATION OF SOIL MEDIUM
The total amount of soil needed for the 4 replicates per treatment level was prepared in one batch, hence 2100 g of dry soil corresponding to 2940 g of wet soil (at a moisture content of 40 % of the dry weight) was made up. Mixing of the soil took place in a mixture.

COMPONENTS OF SOIL MEDIUM (based on % dry weight)
- 10% Sphagnum peat
- 20% Kaolin clay (kaolinite content >30%)
- 69% Industrial quartz sand (> 50% of particles are 50 - 200 µm)
- Approximately 1% calcium carbonate to reach a pH of 6.0 ± 0.5 (in this test 1.48% of CaC03 were added to the artificial soil substrate to reach the desired pH)
- The water content was adjusted to approximately 40% of the dry weight of the soil by the addition of deionized water.

STOCK SOLUTION PREPARATION
Since the test item was not soluble at the desired concentrations in deionized water, 40 g of a stock mixture of test item and fine quartz sand was made up for each test concentration. To reach a test concentration of 95 mg/kg of dry weight of soil, 199.3 mg of the test substance was mixed thoroughly into the sand and then added to the dry blended constituents of the artificial soil. For a test concentration of 171, 309, 556 and 1000 mg/kg dry weight of soil, 358.9, 648.8, 1167.5 and 2099.8 mg of test item, respectively, was pre-mixed with sand and then added to 2060 g (dry weight) of artificial soil. To obtain the desired soil moisture of approximately 40%, for each treatment level 701 mL of deionised water were added to the soil. The moist soil was again thoroughly mixed and then divided up into the test vessels with each test vessel containing approximately 700g of soil (wet weight) corresponding to 500 g dry soil.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Source: Obtained from in-house cultures at the test facility
- Breed: They were bred under standardised conditions in a mixture of peat and cattle manure. Earthworms were not treated against any disease during holding.
- Age at test initiation: Approximate age of 10 months
- Weight at test initiation: Wet mass between 321 and 348 mg
- Feeding: The test organisms were not fed throughout the test period.

PROCEDURE PRIOR TO TEST
Before exposure earthworms were conditioned in the same artificial soil substrate as used in the test for 24 hours. Just prior to test initiation earthworms were quickly washed, weighed individually and the weight of ten animals recorded. Exposure started when test organisms were placed on the soil surface and allowed to enter the soil.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Test temperature:

19.5 - 21.0 °C

pH:

5.7 - 6.0

Moisture:

- Start of test: 39.0 - 39.8% of the dry weight of soil
- End of test: 32.8 - 36.4% of the dry weight of soil

Details on test conditions:

TEST SYSTEM
- Test container: 1.5 L glass bottling jars which were covered by glass lids allowing for gas exchange and minimising evaporation.
- Amount of soil or substrate: Each test container was filled with 700 g of moist soil (500 g dry weight)
- No. of organisms per container: 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
See "Details on preparation and application of test substrate".

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Light intensity: 485 Lux (mean of four measurements: 400, 570, 450, 520)

SOIL QUALITY AND ENVIRONMENTAL MEASUREMENTS
- Soil water content: The soli water concent of each test concentration and the control was measured at the start and the end of the test. An approximate 10 g sample of wet soil was taken and the moisture content determined by drying the soil at 105°C in a vacuum oven. At test termination, a sub-sample of soil (approx. 3 g) from each of the four replicates was taken and pooled for the determination of moisture content of the soil.
- pH: At test initiation, the pH of each batch of soil was measured.
- Temperature: Temperature in the environmental chamber was recorded continuously
- Light intensity: Light intensity was measured at the beginning of the test

EFFECT PARAMETERS MEASURED:
- Weight: At test initiation, the weight of each earthworm was determined individually and the weight of ten earthworms for each replicate recorded.
- Burrowing time: At test initiation and after 7 days of exposure, the time taken by the earthworms to burrow into the soil after initial placing on the soil surface was recorded. Observations of burrowing time were made up to two hours following the positioning of the test organisms on the soil surface.
- Mortality of sign of toxicity: After 7 and 14 days of exposure, observations on mortality and any sublethal effects, such as abnormal behaviour or flaccidity were performed. Dead organisms, if any, were removed from the test soil. At test termination, the weight of the surviving earthworms for each replicate at each test concentration was recorded.

VEHICLE CONTROL PERFORMED: No
- No range - finding test was carried out.

Nominal and measured concentrations:

- Nominal concentration: 0 (negative control), 95, 171, 309, 556 and 1000 mg/kg dry weight of soil

Reference substance (positive control):

yes

Remarks:

Chloracetamide

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

230 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other:

Remarks:

95% C.L.:171 - 309 mg/L dry weight of soil

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

95 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

other: weight

Details on results:

MORTALITY
No mortality of earthworms was observed at day 7 and day 14 of the test, in the control and in test concentrations of 95 and 171 mg test item/kg dry weight of soil. At a test concentration of 309 mg test item/kg dry weight of soil, no mortality was observed after an exposure period of 7 days, however 100% mortality had occurred at this concentration at day 14 of the test. At a test concentration of 556 mg test substance/kg dry weight of soil, 85% mortality of worms was recorded after 7 days of exposure; 100% mortality of worms was observed at day 14 of the test at this test concentration. After 7 days of exposure, 100% mortality of earthworms had occurred at a test concentration of 1000 mg test item/kg dry weight of soil.

SUBLETHAL EFFECTS
No sublethal effects, such as abnormal behaviour or flaccidity or other symptoms were observed in the control and treatment concentrations of 95 and 171 mg of test substance/kg dry weight of soil at day 7 and 14 of the test. At test concentrations of 309 and 556 mg test item/kg dry weight of soil, severe flaccidity of earthworms was recorded at day 7 of the test.

WEIGHT
The average live weight of the earthworms in the control and test concentrations of 95 and 171 mg test substance/kg dry weight of soil, changed to a weight of 97, 91 and 80% of the initial weight during the 14 days of exposure, respectively. The average weight change (in %) in comparison to the controls was significantly different (Dunnett-test; a = 0.05, one-sided, smaller) at a test concentration of 171 mg/kg dry weight of soil. Hence the NOEC (14d) regarding earthworm weight was determined to be 95 mg/kg dry weight of soil.

BURROWING TIME
No increased burrowing time was observed in the control and at test concentrations of 95, 171, 309, 556 and 1000 mg test substance/kg dry weight of soil at the start of the test, all earthworms had burrowed into the soil within 15 minutes of initially placing them on the soil surface. At day 7 of the test, all earthworms in the control and test concentrations of 95 and 171 mg/kg had burrowed into the soil within 15 minutes of initial placing on the soil surface. At a test concentration of 309 and 556 mg test substance/kg dry weight of soil, the remaining earthworms had burrowed into the soil within 30 minutes of initially placing them on the soil surface.

Results with reference substance (positive control):

The quality of the earthworms is checked at least every year by determining the LC 50 (14d) value for the reference item Chloracetamide. The LC 50 was 39 mg/kg (35 - 43 mg/kg 95% confidence limits). The test was carried out in April 2000.

Reported statistics and error estimates:

Statistical analysis of the data for the determination of the LC 50 values at 7 and 14 days of the test was carried out using the statistical package ‘EASY ASSAY Critical Values’ (Ratte H T, 1995a). For the determination of the NOEC in regard to average live weight of test organisms, Dunnett’s multiple means comparison test was performed (Ratte H T, 1995b). Calculations were carried out using Microsoft Excel worksheets, which used an indefinite number of digits for the calculations, however within this report only the rounded values were displayed.

Table 1 Mortality of earthworms after 7 and 14 days 

Nominal conc. (mg/kg of dry weight of soil)	Cumulative mortality
	Day 7		Day 14
	Number of dead animals	%	Number of dead animals	%
Control	0	0	0	0
95	0	0	0	0
171	0	0	0	0
309	0	0	40	100
556	34	85	40	100
1000	40	100	40	100

 

 Table 2. Sublethal effects observed after 7 and 14 days

Nominal conc. (mg/kg of dry weight of soil)	Cumulative mortality
	Day 7			Day 14
	Abnormal behavior	Flaccidity	Others (open wounds)	Abnormal behavior	Flaccidity	Others (open wounds)
Control	0	0	0	0	0	0
95	0	0	0	0	0	0
171	0	0	0	0	0	0
309	0	3	0	††	††	††
556	0†	3†	0†	††	††	††
1000	††	††	††	††	††	††

0:No symptoms

1: Light symptoms

2: Moderate symptoms

3: Severe sympotoms

†: Some worms died

††: All worms died

Table 3. Weight of test organisms at the start and end of the test

Nominal conc. (mg/kg of dry weight of soil)	No. of test vessel	Average live weight of test organisms (mg)		Weight different compared to start of test
		Start of test	End of test	Mg/worm	%	Average (%)
Control	1	332	322	-10	97	97
	2	343	298	-45	87
	3	333	378	45	114
	4	333	304	-29	91
95	5	331	337	6	102	91
	6	331	303	-28	92
	7	325	289	-36	89
	8	331	269	-62	81
171	9	341	284	-57	83	80*
	10	348	261	-87	75
	11	329	254	-75	77
	12	338	281	-57	83
309	13	327	††	††	††	††
	14	328	††	††	††
	15	342	††	††	††
	16	339	††	††	††
556	17	333	††	††	††	††
	18	325	††	††	††
	19	325	††	††	††
	20	321	††	††	††
1000	21	332	††	††	††	††
	22	344	††	††	††
	23	334	††	††	††
	24	348	††	††	††

* Significant (Dunnett-test; α = 0.05, one - sided, smaller) weight change (in %) in comparison to the control)

††: All worms died

Table 4. Burrowing time observed at test start and day 7 of the test

Nominal conc. (mg/kg of dry weight of soil)	No. of test vessel	Burrowing time (time taken by earthworms to burrow into soil after initial placing on the soil surface; observations were made up to 2 hours) in minutes
		Start of test	Day 7
Control	1	Within 15 min	Within 15 min
	2	Within 15 min	Within 15 min
	3	Within 15 min	Within 15 min
	4	Within 15 min	Within 15 min
95	5	Within 15 min	Within 15 min
	6	Within 15 min	Within 15 min
	7	Within 15 min	Within 15 min
	8	Within 15 min	Within 15 min
171	9	Within 15 min	Within 15 min
	10	Within 15 min	Within 15 min
	11	Within 15 min	Within 15 min
	12	Within 15 min	Within 15 min
309	13	Within 15 min	Within 30 min
	14	Within 15 min	Within 30 min
	15	Within 15 min	Within 30 min
	16	Within 15 min	Within 30 min
556	17	Within 15 min	††
	18	Within 15 min	Within 30 min
	19	Within 15 min	††
	20	Within 15 min	Within 30 min
1000	21	Within 15 min	††
	22	Within 15 min	††
	23	Within 15 min	††
	24	Within 15 min	††

††: All worms died

Validity criteria fulfilled:

yes

Remarks:

Less than 10% of earthworms were dead in the control at the end of the test.

Conclusions:

The 14-day LC50 value for the test substance was determined to be 230 mg/kg dry soil (95% confidence interval: 171 - 309 mg/kg dry soil) and the NOEC was determined to be 95 mg/kg dry soil, regarding the earthworm weight.

Executive summary:

The short-term toxicity to terrestrial invertebrates was investigated in a study in accordance with OECD TG 207 and in compliance with GLP using earthworms (Eisenia fetida). Adult earthworms were exposed to through artificial soil as substrate to a series of test concentrations of the test material of 0 (negative control), 95, 171, 309, 556 and 1000 mg/kg dry soil for 14 days. The toxic standard Chloracetamide was tested in a separate study to confirm the normal reaction of the earthworms against pesticides. Throughout the study, the environmental conditions were maintained as recommended in the standard guideline (19.5-21 °C, moisture content 32.8 – 39.8% of dry weight, continuous darkness, no feeding).

 

No mortality of earthworms was observed in the control and at the test concentrations of 95 and 171 mg test item /kg dry weight of soil over the whole study period. At a test concentration of 309 mg test item/kg dry weight of soil, no mortality was observed after an exposure period of 7 days, however 100% mortality had occurred at this concentration at day 14 of the test. At the remaining higher test concentrations, all test animals were dead after 14 days of exposure. No sublethal effects, such as abnormal behaviour or flaccidity or other symptoms were observed in the control and at treatment concentrations of 95 and 171 mg of test substance/kg dry weight of soil at day 7 and 14 of the test. At test concentrations of 309 and 556 mg test item/kg dry weight of soil, severe flaccidity of earthworms was recorded at day 7 of the test. The average live weight of the earthworms in the control and at test concentrations of 95 and 171 mg test substance/kg dry weight of soil, changed to a weight of 97, 91 and 80% of the initial weight during the 14-day exposure period, respectively. The average weight change (in %) in comparison to the controls was significantly different (Dunnett-test; α = 0.05, one-sided, smaller) at a test concentration of 171 mg/kg dry weight of soil. Hence the 14-day NOEC regarding earthworm weight was determined to be 95 mg/kg dry weight of soil.  At test concentrations ≥309 mg test substance/kg dry weight of soil  a significantly increased burrowing time of earthworms placed on the soil surface was observed on day 7. Based on all findings, the 14-day LC50 value for the test substance was estimated to be 230 mg/kg dry soil (95% confidence interval: 171 - 309 mg/kg dry soil) and the NOEC was established at 95 mg/kg dry soil, regarding the earthworm weight.

Description of key information

The 14-d LC50 = 230 mg/kg soil dw, Eisenia fetida, mortality, OECD TG 207, Pfeifle 2000

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

230 mg/kg soil dw

Additional information

The short-term toxicity to terrestrial invertebrates was investigated in a study in accordance with OECD TG 207 and in compliance with GLP using earthworms (Eisenia fetida). Adult earthworms were exposed to through artificial soil as substrate to a series of test concentrations of the test material of 0 (negative control), 95, 171, 309, 556 and 1000 mg/kg dry soil for 14 days. The toxic standard Chloracetamide was tested in a separate study to confirm the normal reaction of the earthworms against pesticides. Throughout the study, the environmental conditions were maintained as recommended in the standard guideline (19.5-21 °C, moisture content 32.8 – 39.8% of dry weight, continuous darkness, no feeding).

No mortality of earthworms was observed in the control and at the test concentrations of 95 and 171 mg test item /kg dry weight of soil over the whole study period. At a test concentration of 309 mg test item/kg dry weight of soil, no mortality was observed after an exposure period of 7 days, however 100% mortality had occurred at this concentration at day 14 of the test. At the remaining higher test concentrations, all test animals were dead after 14 days of exposure. No sublethal effects, such as abnormal behaviour or flaccidity or other symptoms were observed in the control and at treatment concentrations of 95 and 171 mg of test substance/kg dry weight of soil at day 7 and 14 of the test. At test concentrations of 309 and 556 mg test item/kg dry weight of soil, severe flaccidity of earthworms was recorded at day 7 of the test. The average live weight of the earthworms in the control and at test concentrations of 95 and 171 mg test substance/kg dry weight of soil, changed to a weight of 97, 91 and 80% of the initial weight during the 14-day exposure period, respectively. The average weight change (in %) in comparison to the controls was significantly different (Dunnett-test; α = 0.05, one-sided, smaller) at a test concentration of 171 mg/kg dry weight of soil. Hence the 14-day NOEC regarding earthworm weight was determined to be 95 mg/kg dry weight of soil.  At test concentrations ≥309 mg test substance/kg dry weight of soil a significantly increased burrowing time of earthworms placed on the soil surface was observed on day 7. Based on all findings, the 14-day LC50 value for the test substance was estimated to be 230 mg/kg dry soil (95% confidence interval: 171 - 309 mg/kg dry soil) and the NOEC was established at 95 mg/kg dry soil, regarding the earthworm weight.