2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 June 2004 to 19 August 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

Amounts of the test material, 0.540, 2.632 and 10.304 mg, were weighed, dissolved in acetone (20 mL) and dried onto quartz sand (50 g) overnight.
On the day of the test, all treatments were placed under a sample concentrator for two hours and 15 minutes and then homogenised by rolling for 30 minutes. An aliquot of the treated sand was then incorporated into the required soil (600 g dry weight) and mixed (9.5651, 9.8100 and 10.0241 g sand for treatments 0.16, 0.80 and 3.2 mg test material/kg soil respectively). The quantity of water required to bring the soil to 50 % of its maximum water holding capacity (MWHC) was then added and well mixed before being transferred to the test unit. For the control treatment, 10 g of acetone only treated sand was added to the soil (600g dry weight).

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Source: Test site stock
- Age at test initiation: 9 - 10 months (with clitellum)
- Weight at test initiation: 0.3 - 0.6 g
- Medium: mix of artificial soil and compost
- Feed: Pasteurised cow manure

ACCLIMATION
- Acclimation period: 4 days

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

- Soil Temperature: 20.5 - 21.5 °C
- Air Temperature: 20.4 - 21.9 °C

pH:

5.54 - 5.91

Moisture:

Soil moisture on day 0: 38.4 % w/w (equivalent to 50 % WHC).
Soil moisture measured on day 56: 39.2 - 46.2 % w/w (equivalent to 51.1 - 60.2 % WHC).

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 1200 mL plastic boxes (internal dimensions at the soil surface of 15.8 x 10.8 cm) sealed with a plastic lid and perforated with a single hole to allow gaseous exchange.
- Amount of soil or substrate: 600 g dry weight replicates of test material were prepared
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: Four per treatment
- No. of replicates per control: Four per treatment

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
The soil consisted of 10 % by weight sphagnum peat, 20 % by weight kaolin clay, 70 % by weight industrial sand and pasteurised cow manure (air-dried, macerated, sieved to 2 mm) added at 1 g per 100 g mass of soil on a dry weight basis. The dry constituents were blended in the correct proportions and mixed thoroughly.
Following the 28 day observations made of adult worms, the soil was gently replaced, ensuring that no cocoons were visible on the soil surface. A final application of 5 g dry weight of manure was well mixed into the soil substrate to provide food for any juvenile worms during the remainder of the study.

OTHER TEST CONDITIONS
- Photoperiod: 16 hour light/8 hour dark cycle
- Light intensity: 545 lux

EFFECT PARAMETERS MEASURED
After 28 days the contents of each vessel were tipped onto a tray and observations made of behaviour and condition of adult worms.
On day 56 the vessels were placed in a bath of hot water for between 40 minutes and 55 minutes to bring the juveniles to the soil surface to facilitate counting. After removing and counting the juveniles visible on the soil surface the soil was tipped into a tray and thoroughly examined for cocoons and any remaining juveniles. Unhatched cocoons were counted separately. The vessels were re-checked the same day for juveniles.

VEHICLE CONTROL PERFORMED: yes

Nominal and measured concentrations:

0 (control), 0.16, 0.80 and 3.2 mg test material/kg dry soil

Reference substance (positive control):

yes

Remarks:

carbendazim. A reference study was carried out using the reference materials carbendazim (technical material) incorporated into the soil and Carbendazim Flowable applied to the surface of the soil.

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: adult mortality, biomass change, juvenile production

Details on results:

The test material caused no statistically significant effects (P ≤ 0.05) on adult mortality and growth or on juvenile and cocoon production in the earthworm Eisenia fetida at the highest treatment tested, 3.2 mg /kg dry soil (equivalent to 20x the maximum application rate of 120 gae/ha).

Results with reference substance (positive control):

The results were comparable to ISO 11268-2 (1998). The reference material, when incorporated into the soil, should have a significant effect (α = 0.05) at a concentration between 1 and 5 mg carbendazim/kg dry soil. A significant effect in the incorporated test in this study was seen at 1.8 mg carbendazim/kg dry soil for earthworm reproduction. The guideline requires that the reference material, when applied by surface application, should have a significant effect (α = 0.05) at an application rate equivalent of between 250 to 500 g a.i./ha. The significant effect in the surface applied test in this study was seen at 0.358 L Carbendazim Flowable/ha, nominally equivalent to 179 g carbendazim/ha, which was slightly below that stated in the guideline. However, as this was a formulated product, the toxicity of the product was dependent on the total mix of the product and not only the active ingredient. As such, the results were comparable.

Reported statistics and error estimates:

Each set of data was tested for normality (using the Chi-square test and the Shapiro-Wilks test) and for homogeneity of variance (using the Hartley and Bartlett’s tests) using the computer program TOXSTAT 3.4 (1994), before carrying out a suitable parametric multiple comparison procedure. The statistical method selected was based on the pass or failure of the tests described above. The method was then used to locate, where possible, the statistically (P = 0.05) significant no-observed-effect concentration (NOEC) and the lowest-observed-effect concentration (LOEC).

Environmental Parameters

The soil pH range was 5.54 - 5.89 in the controls and 5.57 to 5.91 in the treatments. Soil temperatures ranged from 20.7 to 21.0 °C in the controls and 20.5 to 21.5 °C in the treatments. The air temperature range was 20.4 - 21.9 °C. Soil moisture on day 0 was 38.4 % w/w (equivalent to 50 % WHC). The mean light intensity was 545 lux.

Validity criteria fulfilled:

yes

Conclusions:

The no-observed-effect concentration (NOEC) was determined to be 3.2 mg/kg dry soil for adult mortality, biomass change and for juvenile production. Therefore the lowest-observed-effect concentration (LOEC) was >3.2 mg/kg dry soil for adult mortality, biomass change and juvenile production.

Executive summary:

The toxicity of the test material to earthworms was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline ISO 11268-2.

During the study, three nominal concentrations of test material were tested; 0.16, 0.80 and 3.2 mg/kg dry soil, equivalent to 1x, 5x and 20x the maximum application rate of 120 gae/ha. A water only control was also tested. Each treatment was replicated four times. The test material was incorporated into the test substrate.

The adult worms were removed after 28 days and weighed, leaving the juveniles to develop in the treated soil for a further 28 days. The numbers of juveniles and cocoons present in each vessel were then counted. The treated groups were compared for numbers of juveniles, unhatched cocoons, and change in biomass and mortality of adult worms with the untreated controls.

A reference study was carried out using the reference items, carbendazim (technical material) incorporated into the soil and Carbendazim Flowable applied to the surface of the soil.

Under the conditions of the study the test material caused no statistically significant effects (P ≤ 0.05) on adult mortality and growth or on juvenile and cocoon production in the earthworm Eisenia fetida at the highest treatment tested, 3.2 mg /kg dry soil (equivalent to 20x the maximum application rate of 120 gae/ha).

The no-observed-effect concentration (NOEC) was therefore determined to be 3.2 mg/kg dry soil for adult mortality, biomass change and for juvenile production. Therefore the lowest-observed-effect concentration (LOEC) was >3.2 mg/kg dry soil for adult mortality, biomass change and juvenile production.

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 June 2001 to 28 August 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

Appropriate amounts of test material were mixed into deionized water and then added to dry soil in each test vessel and thoroughly mixed.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworms

ACCLIMATION
- Acclimation period: 3 days
- Acclimation conditions: Same as test. The temperature during acclimation ranged from 21.7 to 21.9°C. The worms, which had well developed clitella, were not fed during acclimation or testing. Before the test, the worms were quickly and gently washed with deionised water, blotted on paper towels, and weighed.
- Health during acclimation (any mortality observed): During acclimation worms were not treated for disease and they were free of apparent disease, injuries, and abnormalities at the beginning of the test. No mortality was observed during the acclimation period.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

14 d

Test temperature:

20 ± 2°C

Moisture:

Deionised water was used to achieve a nominal moisture content of approximately 30 % (30 % was determined to be the highest moisture content that did not cause excess moisture to puddle on the surface of the soil).

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 0.5 gallon glass jars
- Amount of soil or substrate: 1.0 kg of hydrated artificial soil per jar
- No. of organisms per treatment: 40 (10 per replicate)
- Loading rate: 3.3 g worm per kg wet soil

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
Soil (Lot # 081301) was formulated by combining industrial sand, kaolin clay, and finely ground sphagnum peat in a 70:20:10 ratio by weight and the pH was adjusted to 6.0 ± 0.5 by the addition of calcium carbonate. At the beginning of the test the control soil had a moisture content of 30 % and a pH of 6.5. At the end of the test the soil moisture content was 28 %. A representative sample of artificial soil was characterised for pesticides and PCBs. That soil sample contained <0.05 mg/kg organophosphorus pesticides, PCBs, and organochlorine pesticides.

OTHER TEST CONDITIONS
- Photoperiod: A 24 hour light and 0 hour dark photoperiod was maintained throughout the test
- Light intensity: ca. 780 lux

EFFECT PARAMETERS MEASURED
The number of surviving organisms and the occurrence of sublethal effects were determined visually and recorded initially and after 7 and 14 days. The time required to burrow was recorded after 0, 7, and 14 days, and the average wet weight of surviving worms was determined at the beginning of the test and after 14 days.
Soil temperature was measured and recorded daily in each test vessel and the temperature in one control vessel was recorded continuously during the test.

VEHICLE CONTROL PERFORMED: yes

PRELIMINARY TEST
A range-finding test was conducted for 14 days. Results of this test were inconclusive and the test was repeated. A second range-finding test was conducted for 14 days. Nominal concentrations of test material were 0 (control) and 1000 mg/kg (dry weight) and the test material was mixed into deionised water and then added to dry soil. After 14 days there was 100 % survival in the control and at 1000 mg/kg.

Nominal and measured concentrations:

0 (control) and 1000 mg/kg (dry weight)

Reference substance (positive control):

yes

Remarks:

2-chloracetamide

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, sub-lethal effects, weight loss

Details on results:

After 14 days during the range-finding toxicity test there was 100 % survival in the control and at 1000 mg/kg. A 14 day soil toxicity test was conducted with the reference toxicant 2-chloracetamide, resulting in an LC50 of 17 mg/kg, wet weight. This value is within the range normally obtained in the test laboratory.
Nominal concentrations of test material in soil were 0 (control) and 1000 mg/kg. Temperature during the test ranged from 20.1 to 21.8 °C.
An average of 98 % survival occurred and no sublethal effects were observed in the control exposure. The average weight gain of control worms was 3.4 %. All surviving worms exposed to the control and 1000 mg/kg test material burrowed into the soil within 10 minutes on days 0, 7, and 14.

Reported statistics and error estimates:

The 7 and 14 day LC50 values could not be calculated because 98 % survival occurred at the single tested concentration. The NOEC was determined at 14 days using combined survival and sublethal effect data, time to burrow, and weight change data. Survival and sublethal effect data did not warrant statistical analysis because identical survival occurred in the control and treatment, and no sublethal effects were observed. Since all worms burrowed within 10 minutes, statistical analysis was not warranted. The weight change data from the treatment were compared to the control data using a parametric unpaired “t” test.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of worms to the test material resulted in 7 and 14 day median lethal concentrations (LC50s) greater than 1000 mg/kg. The 14 day no observed effect concentration (NOEC) was 1000 mg/kg, based on survival and sublethal effects, time to burrow, and weight loss.

Executive summary:

The toxicity of the test material to earthworms was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 207.

During the study, worms were exposed to 0 (control) or 1000 mg/kg test material in soil, at a temperature of 20 ± 2 °C. The test media was artificial soil comprised of approximately 70 % sand, 20 % kaolin clay, and 10 % finely ground sphagnum peat moss. Test material was added to deionised water which was then transferred to dry artificial soil in each test vessel and thoroughly mixed. The initial moisture content was 30 % and the final moisture content was approximately 28 %.

Forty worms (10 per replicate) were exposed to the following nominal concentrations of test material, determined on a dry weight basis: 0 (control) and 1000 mg/kg. Exposure of worms to test material resulted in 7 and 14 day median lethal concentrations (LC50s) greater than 1000 mg/kg, based on survival. The 14 day no observed effect concentration (NOEC) in this test was 1000 mg/kg based on survival, sublethal effects, and weight loss.

Description of key information

SHORT-TERM
LC50 = >1000 mg/kg soil dw, OECD 207, Ward & Boeri (2001)
LONG-TERM
NOEC (adult mortality, biomass change, juvenile production) = 3.2 mg/kg soil dw, ISO 11268-2, Davies (2004)

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

1 000 mg/kg soil dw

Long-term EC10, LC10 or NOEC for soil macroorganisms:

3.2 mg/kg soil dw

Additional information

Two studies, investigating the toxicity of the substance to earthworms are available. Both studies were conducted under GLP conditions and in accordance with standardised guidelines. Both studies were assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997).

In the study reported by Ward & Boeri (2001) the toxicity of the test material to earthworms was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 207.

During the study, worms were exposed to 0 (control) or 1000 mg/kg test material in soil, at a temperature of 20 ± 2 °C. The test media was artificial soil comprised of approximately 70 % sand, 20 % kaolin clay, and 10 % finely ground sphagnum peat moss. Test material was added to deionised water which was then transferred to dry artificial soil in each test vessel and thoroughly mixed. The initial moisture content was 30 % and the final moisture content was approximately 28 %.

Forty worms (10 per replicate) were exposed to the following nominal concentrations of test material, determined on a dry weight basis: 0 (control) and 1000 mg/kg. Exposure of worms to test material resulted in 7 and 14 day median lethal concentrations (LC50s) greater than 1000 mg/kg, based on survival. The 14 day no observed effect concentration (NOEC) in this test was 1000 mg/kg based on survival, sublethal effects, and weight loss.

In the study reported by Davies (2004) the toxicity of the test material to earthworms was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline ISO 11268-2.

During the study, three nominal concentrations of test material were tested; 0.16, 0.80 and 3.2 mg/kg dry soil, equivalent to 1x, 5x and 20x the maximum application rate of 120 gae/ha. A water only control was also tested. Each treatment was replicated four times. The test material was incorporated into the test substrate.

The adult worms were removed after 28 days and weighed, leaving the juveniles to develop in the treated soil for a further 28 days. The numbers of juveniles and cocoons present in each vessel were then counted. The treated groups were compared for numbers of juveniles, unhatched cocoons, and change in biomass and mortality of adult worms with the untreated controls.

A reference study was carried out using the reference items, carbendazim (technical material) incorporated into the soil and Carbendazim Flowable applied to the surface of the soil.

Under the conditions of the study the test material caused no statistically significant effects (P ≤ 0.05) on adult mortality and growth or on juvenile and cocoon production in the earthworm Eisenia fetida at the highest treatment tested, 3.2 mg /kg dry soil (equivalent to 20x the maximum application rate of 120 gae/ha).

The no-observed-effect concentration (NOEC) was therefore determined to be 3.2 mg/kg dry soil for adult mortality, biomass change and for juvenile production. Therefore the lowest-observed-effect concentration (LOEC) was >3.2 mg/kg dry soil for adult mortality, biomass change and juvenile production.