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Toxicity to soil macroorganisms except arthropods

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Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
12 February 1997 and 20 May 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Deviations:
yes
Remarks:
.These deviations did not affect the outcome of the study and no other deviations from the protocol were made during the study.
Qualifier:
according to guideline
Guideline:
EU Method C.8 (Toxicity for Earthworms: Artificial Soil Test)
Deviations:
yes
Remarks:
.These deviations did not affect the outcome of the study and no other deviations from the protocol were made during the study.
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
yes
Details on preparation and application of test substrate:
A range-finding test was conducted for 14 days from February 24 to March 10, 1997. Test substance was mixed with deionized water to formulate stock solutions which were then added to test vessels containing soil. Nominal concentrations of NBPT (the active ingredient of Agrota~) used for this test were 0 (control), 0.010, 0.10, 1.0, 10, 100, and 1,000 mglkg. The soil for this test had a 25% moisture content. After 14 days of exposure there was 100% survival in the control vessels and at 0.010, 1.0, 10, and 100 mg a.i./kg, 90% survival at 0.10 mg a.i./kg, and 0% survival at 1,000 mg aJ./kg. The average weight change of worms at each tested concentration was: control = -20%, 0.010 mg a.i./kg = -16%,0.10 mg a.i./kg = -20%, 1.0 mg a.i./kg = -17%, 10 mg a.i./kg = -20, and 100 mg a.i./kg = -15%.

The definitive toxicity test was conducted for 14 days from March 11 to 25, 1997, according to T.R. Wilbury Study Protocol 1225-TS (Acute Toxicity of Agrotain to the Earthworm, Eisenia foetida), which was signed by the study director on February 12, 1997. It is based on procedures of the GEeD (1984) and the U.S. FDA (1984). This test was conducted at 20 ± 2°C with five concentrations oftest substance and a control.

Separate stock solutions were prepared for each of the five test concentrations and the control. Each stock solution was prepared by adding the appropriate amount of test substance to a glass, class-A volumetric flask, bringing the final volume to 1,000 ml with deionized water, then mixing. For each test concentration, 250 g of stock solution was added to each one of four test vessels, each vessel containing 750 g of dry soil. The final weight in each test vessel was 1 kg with a final moisture content of approximately 25%. Nominal concentrations of the active ingredient of the test substance were 0 mg/kg (control), 130, 220, 360, 600, and 1,000 mglkg, wet weight. The dry weight concentrations were then calculated based on the assumption that the wet soil contained only the 25% water that was added to the dry soil mixture at the start ofthe toxicity test.

Ten worms were indiscriminately placed into each of four replicates for each treatment. Test vessels were 3.8 liter glass jars that contained 1 kg of soil (loading rate was approximately 3.6 g worm per kg soil). These vessels were randomly arranged for the 14 day test (a random numbers table was used to choose the location of each test vessel) and were covered with ventilated closures. A 24 hour light and 0 hour dark photoperiod was maintained throughout the test at an average light intensity of 500 lux. Worms were not fed during the test.

The number of surviving organisms and the occurrence of sublethal effects were detennined visually and recorded initially and after 7 and 14 days, the time required to burrow was recorded initially and after 7 days, and the average wet weight of live worms was determined initially 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.
Test organisms (species):
Eisenia fetida
Animal group:
nematods
Details on test organisms:
TEST ORGANISM
- Common name:
Earthworm

- Source:
They were purchased
From a commercial supplier (Carolina Biological Supply Company, Burlington, North Carolina)

- Age at test initiation (mean and range, SD):
Not reported

- Weight at test initiation (mean and range, SD):
Not reported. The weight of each earthworm was recorded on Day 0 before addition to the test or control vessels.

ACCLIMATION
- Acclimation period:
4 days

- Acclimation conditions (same as test or not):
Artificial soil without test material.

- Health during acclimation (any mortality observed):
No mortality was obsetved
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
yes
Total exposure duration:
14 d
Post exposure observation period:
The number of surviving organisms and the occurrence of sublethal effects were detennined visually and recorded initially and after 7 and 14 days, the time required to burrow was recorded initially and after 7 days, and the average wet weight of live worms was determined initially and after 14 days.
Test temperature:
20 ± 2°C
Soil temperature was measured and recorded daily in each test vessel, and the temperature in one control vessel was recorded continuously during the test.
pH:
6.0 ± 0.5
Moisture:
Moisture content of approximately 25%.
Details on test conditions:
A range-finding test was conducted for 14 days from February 24 to March 10, 1997. Test substance was mixed with deionized water to formulate stock solutions which were then added to test vessels containing soil. Nominal concentrations of NBPT (the active ingredient of Agrota~) used for this test were 0 (control), 0.010, 0.10, 1.0, 10, 100, and 1,000 mglkg. The soil for this test had a 25% moisture content. After 14 days of exposure there was 100% survival in the control vessels and at 0.010, 1.0, 10, and 100 mg a.i./kg, 90% survival at 0.10 mg a.i./kg, and 0% survival at 1,000 mg aJ./kg. The average weight change of worms at each tested concentration was: control = -20%, 0.010 mg a.i./kg = -16%,0.10 mg a.i./kg = -20%, 1.0 mg a.i./kg = -17%, 10 mg a.i./kg = -20, and 100 mg a.i./kg = -15%.

The definitive toxicity test was conducted for 14 days from March 11 to 25, 1997, according to T.R. Wilbury Study Protocol 1225-TS (Acute Toxicity of Agrotain to the Earthworm, Eisenia foetida), which was signed by the study director on February 12, 1997. It is based on procedures of the OECD (1984) and the U.S. FDA (1984). This test was conducted at 20 ± 2°C with five concentrations oftest substance and a control.

Separate stock solutions were prepared for each of the five test concentrations and the control. Each stock solution was prepared by adding the appropriate amount of test substance to a glass, class-A volumetric flask, bringing the final volume to 1,000 rn1 with deionized water, then mixing. For each test concentration, 250 g of stock solution was added to each one of four test vessels, each vessel containing 750 g of dry soil. The final weight in each test vessel was 1 kg with a final moisture content of approximately 25%. Nominal concentrations of the active ingredient of the test substance were 0 mg/kg (control), 130, 220, 360, 600, and 1,000 mg/kg, wet weight. The dry weight concentrations were then calculated based on the assumption that the wet soil contained only the 25% water that was added to the dry soil mixture at the start ofthe toxicity test.

Ten worms were indiscriminately placed into each of four replicates for each treatment. Test vessels were 3.8 liter glass jars that contained 1 kg of soil (loading rate was approximately 3.6 g worm per kg soil). These vessels were randomly arranged for the 14 day test (a random numbers table was used to choose the location of each test vessel) and were covered with ventilated closures. A 24 hour light and 0 hour dark photoperiod was maintained throughout the test at an average light intensity of 500 lux. Worms were not fed during the test.

The number of surviving organisms and the occurrence of sublethal effects were detennined visually and recorded initially and after 7 and 14 days, the time required to burrow was recorded initially and after 7 days, and the average wet weight of live worms was determined initially 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.
Nominal and measured concentrations:
Range-finding
Nominal concentrations of NBPT (the active ingredient of Agrota~) used for this test were 0 (control), 0.010, 0.10, 1.0, 10, 100, and 1,000 mg/kg.

Definitve Test:
Nominal concentrations of the active ingredient of the test substance were:
0 mg/kg (control), 130, 220, 360, 600, and 1,000 mg/kg, wet weight.
Reference substance (positive control):
yes
Remarks:
2-chloracetamide
Duration:
7 d
Dose descriptor:
LC50
Effect conc.:
860 other: mg a.i/kg
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Based on survival, sublethal effects, time to burrow, and weight loss relative to the control.
Remarks on result:
other: 95% confidence interval = 760 to 900 mg a.i/kg.
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
830 other: mg a.i/kg
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Based on survival, sublethal effects, time to burrow, and weight loss relative to the control.
Remarks on result:
other: 95% confidence interval = 760 to 900 mg a.i/kg.
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
600 other: mg a.i/kg
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Based on survival, sublethal effects, time to burrow, and weight loss relative to the control.
Remarks on result:
other: 95% CL not stated
Details on results:
Temperature during the test ranged from 19.0 to 20.9°C. One hundred percent survival occurred and no sublethal effects were observed in the control exposure. Worms exposed to the control and all concentrations of test substance burrowed into the soil in less than 10 minutes on days 0 and 7. A14-day soil toxicity test was conducted from September 26 to October 10, 1996 with the reference toxicant 2-chloracetamide, resulting in an LC50 of 10 mg/kg.

Survival of worms exposed to 1,000 mg a.i./kg averaged 20% and survival of worms exposed to each of the four lower tested concentrations of Agrotain averaged 92-100% after 14 days. The time to burrow was less than ten minutes in each test vessel containing live organisms on days 0 and 7. Control worms lost an average of 9.2% of their initial weight during the 14 day test, surviving worms exposed to Agrotain (130 and 1,000 mg a.i./kg) lost an average of 1.1 and 23% of their initial weight, respectively, and surviving worms exposed to 220, 360, and 600 mg a.i./kg gained an average of 2.2%, 4.0%, and 0.60% of their initial weight, respectively.

The 7 day LC50 for worms exposed to Agrotain is 860 mg a.i./kg (95% confidence interval = 600 to 1,000 mg a.i./kg), and the 14 day LC50 is 830 mg a.i./kg (95% confidence interval = 760 to 900 mg a.i./kg). The 7-day and 14-day LC50 values were equivalent to 1,100 mg a.i./kg when calculated on a dry weight basis. The NOEC is 600 mg a.i./kg (equivalent to 800 mg a.i./kg, calculated dry weight), based on survival, sublethal effects, time to burrow, and weight loss relative to the control.
Results with reference substance (positive control):
- Results with reference substance valid?
Yes

- Relevant effect levels:
A14-day soil toxicity test was conducted from September 26 to October 10, 1996 with the reference toxicant 2-chloracetamide, resulting in an LC50 of 10 mg/kg.
Reported statistics and error estimates:
The results of the toxicity test were interpreted using standard statistical techniques (Stephan, 1983). The 7 day median lethal concentration (LC50) was
calculated using the binomial/nonlinear interpolation method, and the 14 day LC50 was calculated using the probit method. The NOEC was determined using percent survival. The data were transformed (arc sin square root), and means from the five treatment exposures were compared to control data using a William'stest. These data were demonstrated to be non-normally distributed with a Chi-square test and variances were demonstrated to be heterogeneous with a Bartlett's test ( α = 0.05). Because all worms burrowed within ten minutes, and worms exposed to concentrations of Agrot~ that did not cause a significant decrease in survival lost less weight than the controls and did not exhibit sublethal effects, these data were not statistically analyzed.
Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test material, to the earthworm Eisenia foetida, in an artificial soil test has been investigated. The 7 day LC50 for worms exposed to Agrotain is 860 mg a.i./kg (95% confidence interval = 600 to 1,000 mg a.i./kg), and the 14 day LC50 is 830 mg a.i./kg (95% confidence interval = 760 to 900 mg a.i./kg). The 7-day and 14-day LC50 values were equivalent to 1,100 mg a.i./kg when calculated on a dry weight basis. The NOEC is 600 mg a.i./kg (equivalent to 800 mg a.i./kg, calculated dry weight), based on survival, sublethal effects, time to burrow, and weight loss relative to the control.
Executive summary:

A study was performed to assess the acute toxicity of the test material to the earthworm (Eisenia foetida) in an artificial soil. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 207 "Earthworm, Acute Toxicity Tests" and EEC Commission Directive 87/302/EEC.

The test substance, Agrotain [reported purity 25% active ingredient; N-(n-butyl) thiophosphoric triamide (NBPT)] was supplied by the sponsor.

The test was performed with five concentrations of test substance and a control at 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 substance was mixed with deionized water then added to dry artificial soil in each test vessel. The final moisture content in each test vessel was approximately 25%. All nominal calculations are based on milligrams of active ingredient per kilogram of wet soil, unless otherwise noted.

Forty worms per concentration (10 worms per replicate, 4 replicates per concentration) were exposed to the following nominal concentrations of Agrotain (control), 130,220,360, 600, and 1,000 mg a.i./kg. Exposure ofwonns to Agrot~ resulted in a 7 day median lethal concentration (LC50) of 860 mg a.i./kg (equivalent to 1,100 mg a.i./kg, calculated dry weight), and a 14 day LC50 of 830 mg a.i./kg (equivalent to 1,100 mg a.i./kg, calculated dry weight).

The acute toxicity of the test material, to the earthworm Eisenia foetida, in an artificial soil test has been investigated. The 7 day LC50 for worms exposed to Agrotain is 860 mg a.i./kg (95% confidence interval = 600 to 1,000 mg a.i./kg), and the 14 day LC50 is 830 mg a.i./kg (95% confidence interval = 760 to 900 mg a.i./kg). The 7-day and 14-day LC50 values were equivalent to 1,100 mg a.i./kg when calculated on a dry weight basis. The NOEC is 600 mg a.i./kg (equivalent to 800 mg a.i./kg, calculated dry weight), based on survival, sublethal effects, time to burrow, and weight loss relative to the control.

Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2015-09-11 to 2015-11-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with standard OECD guideline under GLP with well documented report
Qualifier:
according to guideline
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Deviations:
yes
Remarks:
Minor: continuous lighting
GLP compliance:
yes (incl. QA statement)
Remarks:
2015-06-04
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
Immediately before the start of the experiment, test item stock solution was prepared by diluting an amount of 12752.7 mg of test item in 2000 mL deionised water. The stock solution was then further diluted according to the Table 1 in "Any other materials and methods incl. tables" field below in order to prepare the test item solutions.

Immediately before the start of the test, the eight test item solutions were then mixed with the artificial soil so that the moisture of the soil was approximately 60 % of the maximum WHC which corresponded about 21.7 % of the soil dry weight.
Care was taken to avoid any contamination of the control. The test item concentrations were applied in increasing order, thus minimizing the risk of contamination.
A homogenous distribution of the test item in the artificial soil was obtained by thorough mixing. In order to obtain a better distribution of the test solutions in the soil, the test substrate was prepared by mixing the amount of soil needed for two replicates (1258.0 g dry weight) in one step.

Untreated Control: Deionised water mixed into the test substrate.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
Taxonomic Group: Earthworms (Annelida: Oligochaeta)
Species: Eisenia fetida (Savigny 1826)
Life Stage: adults, adequately fed, healthy, disease-free colonies
Age: about 5 months (not differing in age by more than 4 weeks) with clitellum
Body Weight: 273 - 599 mg, including gut contents
Origin: BASF SE Crop Protection Division, Limburgerhof
Strain code: VT BASF 05 09 138 – 139 – 140 (in CiToxLAB Hungary Ltd.) Bred under standardised conditions (according to OECD 207 guideline) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd., Veszprém-Szabadságpuszta
Acclimatisation: 1 day, in artificial soil, under test environmental conditions (day–1 – day 0)
Number of animals: There were 40 earthworms at each concentration divided into 4 replicates (10 worms/replicate) while 80 earthworms were used in the untreated control divided into 8 replicates (10 worms/replicate)
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
28 d
Post exposure observation period:
28 days
Test temperature:
The temperature was recorded (using a min/max thermometer) within the climate chamber on weekdays during the test. The temperature ranged from 19.2 °C to 20.9°C.
pH:
The pH values of the treated artificial soil were determined at the beginning (Day 0: 5.45 - 6.25) and at the end (Day 56: 6.18 - 6.42) of the test. The determination of pH value was done according to ISO 10390.

Moisture:
At the start of the test the moisture content of the test medium was adjusted to about 60 % of the maximum WHC which corresponded about 21.7 % of the soil dry weight.
Soil moisture content was determined in one replicate at three concentration levels at the beginning and at the end of the test by SARTORIUS Moisture Analyzer.
The average water content was 26.87 % and 25.95% of soil dry weight at the beginning and at the end of the study, respectively.
Details on test conditions:
TEST SYSTEM
- Test container (material, size): plastic containers (with ~1L volume and ~200 cm2 area), filled with the moist artificial soil These plastic containers were covered with transparent and punctured lid to allow gas exchange.
- Amount of soil or substrate: (the amount of the soil: 765.9 g/test container corresponded to 600 g soil dry weight).
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per vehicle control: 8
- Pre-exposure period: the earthworms were conditioned for about 24 hours before treatment in the test substrate. On day 0, after washing and drying gently, the individual weight of each worm was determined, and then worms were exposed distributed randomly to the treated artificial soil.
- Exposure period: the earthworms were housed during 28 days in plastic containers filled with 765.9 g/test container moist artificial soil (corresponding to 600 g soil dry weight) covered with transparent and punctured lid to allow gas exchange.
- Incubation period: 28 days

FEEDING
One day after the start of the exposure period the earthworms were fed with approximately 5 g air-dried finely ground cow manure. It was spread on the soil surface of the containers and moistened with the amount of missing water. Food was provided once a week during the exposition period, on days: 1, 7, 14, 21 and 28. The amounts of cow manure added depended on the amounts of food consumed by the worms as assess visually in comparison with the controls. The water content of the test substrate was controlled weekly during the test by weighing the containers and replenishing the missing water.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
Artificial soil was prepared, as follows:
- 10 % sphagnum peat (as close to pH 5.5-6.0 as possible, no visible plant remains, finely ground, dried to measured moisture content)
- 20 % kaolin clay (with kaolinite content above 30 %)
- 70 % industrial sand (fine sand is dominant with more than 50 % of the particles between 50 and 200 μm)
The air-dry constituents were blended in the correct proportions and mixed thoroughly with deionised water. The final water content was equivalent to about 60 % of the maximum Water Holding Capacity (WHC) determinated according to ANNEX 2 OF OECD 222. The maximal water holding capacity of the artificial soil was 46.09 g water / 100 g soil dry weight.
The moistened soil was divided in test containers corresponding with the number of test concentrations and controls.The water content of the test substrate was controlled weekly during the test by weighing the containers and replenishing the missing water.

TEST CONCENTRATIONS
The concentrations of NBPT investigated in this test were based on the results of the earthworm acute toxicity test performed by the same laboratory and showing an EC50= 1786.26 mg/kg soil dw and a NOEC= 750 mg/kg Soil dw (Study code: 15/161-125G from K. Sipos ,2015) as requested by the Sponsor.
Immediately before the start of the test, the eight test item solutions were then mixed with the artificial soil so that the moisture of the soil was approximately 60 % of the maximum WHC. The test item concentrations were applied in increasing order, thus minimizing the risk of contamination.
A homogenous distribution of the test item in the artificial soil was obtained by thorough mixing. In order to obtain a better distribution of the test solutions in the soil, the test substrate was prepared by mixing the amount of soil needed for two replicates (1258.0 g dry weight) in one step.

OTHER TEST CONDITIONS
- Photoperiod: Continuous lighting
- Light intensity: 583 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortality:
Mortality of the earthworms was assessed between days 1 and 28 of the experiment. On day 28 of the experiment the adult worms were removed from the soil and the number of living adult worms were recorded. Mortality was assessed by emptying the test medium from each replicate onto a suitable tray and sorting worms from the medium. Earthworms were considered dead when they failed to respond to gentle stimulation at the front end. Due to rapid decomposition under test conditions, missing earthworms were considered dead.

Clinical Symptoms:
During the exposure period (days 0-28) symptoms of earthworms were observed without disturbing the soil.

Biomass:
The weight of the live adult worms in every test container was determined on days 0 and 28 of the experiment after they had been removed from the substrate and been gently washed and dried.

Feed consumption:
The feed consumed was estimated with visual checking. The quantity of food applied was recorded for each container on days: 1, 7, 14, 21, 28.

Number of juvenile worms (reproduction):
On day 56 of the experiment the juvenile worms hatched from cocoons were removed from the soils using water bath at a temperature 50-60oC and counted.


Nominal and measured concentrations:
0 (control), 87.79, 131.69, 197.53, 296.30, 444.44, 666.67, 1000 and 1500 mg/kg soil dry weight
Reference substance (positive control):
yes
Remarks:
2-Chloroacetamide
Duration:
56 d
Dose descriptor:
EC50
Effect conc.:
595.53 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
number of juveniles
Remarks on result:
other: 550.36 – 644.41 mg/kg soil dry weight
Duration:
56 d
Dose descriptor:
other: NOAEC
Effect conc.:
197.53 mg/kg soil ww
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
number of juveniles
Duration:
56 d
Dose descriptor:
other: LOAEC
Effect conc.:
296.3 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
number of juveniles
Duration:
28 d
Dose descriptor:
LC50
Effect conc.:
> 1 500 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
1 500 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Mortality at end of exposure period:
No mortality of adult (parent) worms was noted in the test item groups of 87.79, 296.30, 444.44 and 1000 mg/kg soil dry weight after 28 days of exposure. One dead earthworm (mortality of 1.25 %) was noted in the control and in the 131.69 and 1500 mg/kg soil dry weight groups. Two animals (2.5 % mortality) were dead in the 197.53 mg/kg test item group, while three dead earthworm(s) were recorded (mortality rate of 7.5 %) in the test item treated group of 666.67 mg/kg soil dry weight during the experiment. These mortalities were not considered to be due to a toxic effect of the test substance since they were independent from the treated level (see table for details data).

- Clinical symptoms:
During the exposure period (days 0-28) no symptoms of earthworms were observed. On day 28 of the experiment the surviving adult worms in each test container were removed from the soil, and no unusual symptoms were observed.

- Changes in body weigth of live adults (% of initial weight) at end of exposure period:
There was a decrease in earthworm biomass in the untreated control (-12.91 %) and, at a lower extend, in all test item concentrations. This resulted that the biomass was found significantly increased at the tested concentrations of 666.67 and 1000 mg/kg soil dry weight by comparison with the control (Bonferroni t-test, p=0.05). However, it is considered that the observed decreases in biomass in the samples are not related with the toxicity of NBPT since they were independent from the treated level (see table for details data).

-Food consumption:
The feed consumption was lower when compared to the control at the highest concentration of 1500 mg/kg soil dry weight (see table for details data)

- Reproduction (Number of offspring produced):
The mean number of juveniles was significantly lower compared to the control group at the four highest test item concentrations: 444.44, 666.67, 1000 and 1500 mg/kg soil dw. Consequently it is considered that the number of juveniles is decreasing with the increase of NBPT concentration and no juveniles were observed at the highest test item concentration of 1500 mg/kg soil dw indicating that no reproduction occurs in this group.
Results with reference substance (positive control):
As toxic reference item Carbendazim (a.i. content 99 %) was used. At least once a year a study with the reference item is performed to verify the sensitivity of the earthworms and the precision of the test procedure. In the most recent test with the reference item Carbendazim performed at CiToxLAB Hungary Ltd., (ran between 20 February and 17 April 2015, Study Code: 15/027-211G) the EC50 after 56 days was determined to be 0.86 mg Carbendazim /kg soil dry weight (95 % confidence limits:
0.79 – 0.93 mg a.i./kg soil dry weight).
Reported statistics and error estimates:
For growth, worm mean weights and standard deviations were calculated for each treatment at the start and at the end of the exposure period (28d) using Excel for Windows software.
For fecundity, the number of juveniles mean values and standard deviations were calculated for each treatment at the end of the incubation period (56 days) using Excel for Windows software.
For mortality, the LC50 value of the test item could not be calculated due to the slight effects observed.
For reproduction (number of juveniles) , the EC50 value of the test item with its 95% confidence limits were calculated using a Probit analysis by TOXSTAT software.
Statistical analysis of mortality, reproduction (number of juveniles) and worm biomass data's were assessed by analysis of variance (ANOVA) and Bonferroni t-test (p = 0.05) by TOXSTAT software.
.

Earthworm Mortality after 28 Days at Different Concentrations of NBPT

(N-(n-butyl) thiophosphoric triamide )

 

Concentration of
Test Item
[mg/kg soil dry weight]

Number of dead worms / replicates

Mean [%]

SD

1

2

3

4

(5)

(6)

(7)

(8)

Control

0

1

0

0

1.25

3.5

0

0

0

0

87.79

0

0

0

0

0.0n.s

0.0

131.69

0

0

1

0

2.5n.s

5.0

197.53

0

0

1

1

5.0n.s

5.8

296.30

0

0

0

0

0.0n.s

0.0

444.44

0

0

0

0

0.0n.s

0.0

SD          :              Standard deviation

n.s.         :              statistically not significant compared to the control (Bonferroni t-test, α =0.05)

Weight Changes [%] of the Parent Earthworms at Different Concentrations of NBPT (N-(n-butyl) thiophosphoric triamide )

Concentration ofTest Item[mg/kg soil dry weight]

Weight change [%]

Mean [%]

SD

1

2

3

4

(5)

(6)

(7)

(8)

Control

-16.31

-17.27

-10.44

-14.35

-12.91

3.25

-11.11

-7.67

-14.55

-11.61

87.79

-7.41

-14.15

-8.04

-6.26

-8.97n.s

3.54

131.69

-13.30

-10.07

-1.77

-14.65

-9.95n.s

5.78

197.53

-5.56

-11.30

-3.15

-11.61

-7.91n.s

4.22

296.30

-10.38

-8.70

-5.99

-4.14

-7.30n.s

2.78

444.44

-5.01

-6.34

-3.74

-7.89

-5.74n.s

1.78

666.67

-6.85

-2.62

-2.59

-4.93

-4.25*

2.05

1000

-4.50

-4.02

-3.59

2.16

-2.49*

3.12

1500

-0.20

-12.86

-17.30

-9.88

-10.06n.s

7.25

SD:        Standard deviation

n.s.         :              statistically not significant compared to the control (Bonferroni t-test, α =0.05)

*             :              statistically significant differences compared to the control (Bonferroni t-test, α =0.05)

Total Amount of Feed (g Dry Cow Manure) Added per concentration [Average of Four Replicates (Eight Replicates in case of the Control)] during the Course of the Test

Concentration of
Test Item
[mg/kg soil dry weight]

Total amount of dry cow manure added per variant (average of four or eight replicates)

[g]

Control

25.00

87.79

25.00

131.69

25.00

197.53

25.00

296.30

25.00

444.44

25.00

666.67

24.00

1000

21.00

1500

16.00

 

Number of Juveniles measured at Different Concentrations of NBPT

Concentration of
Test Item
[mg/kg soil dry weight]

Number of juveniles

Mean

SD

1 (5)

2 (6)

3 (7)

4 (8)

Control

165

184

175

198

187.38

20.32

185

210

163

219

87.79

182

177

203

179

185.25n.s

12.01

131.69

162

197

173

185

179.25n.s

15.11

197.53

196

188

178

147

177.25n.s

21.47

296.30

166

184

148

161

164.75n.s

14.91

444.44

142

126

152

143

140.75*

10.81

666.67

104

82

102

66

88.50*

17.99

1000

37

50

42

48

44.25*

5.91

1500

0

0

0

0

0.00*

0.00

SD          :              Standard deviation

n.s.         :              statistically not significant compared to the control (Bonferroni t-test, α=0.05)

*             :              statistically significant differences compared to the control (Bonferroni t-test, α =0.05)

Validity criteria fulfilled:
yes
Remarks:
Mortality was 1.25 % and the mean reproduction was 187.38 juveniles per container in the control. The coefficient of variation of reproduction was 10.84 %.
Conclusions:
The test item NBPT (N-(n-butyl) thiophosphoric triamide) doesn’t have an effect on the mortality or the biomass of the adult worms (after 28 days), but have an adverse effect on the reproduction of earthworms (Eisenia fetida) based on the number of juveniles observed after 56 days

For Eisenia fetida adult mortality and biomass development):
LC50 (28 day): > 1500 mg Test Item/kg soil dry weight
NOEC (28 day): 1500 mg/kg soil dry weight

For reproduction of Eisenia fedita based on the number of juveniles:
EC50 (56 day): 595.53 mg/kg soil dry weight (95 % confidence limits: 550.36 – 644.41 mg/kg soil dry weight)
NOAEC (56 day): 197.53 mg Test Item/kg soil dry weight
LOAEC (56 day): 296.30 mg Test Item /kg soil dry weight
Executive summary:

A reliable chronic study was performed to assess the effects of NBPT (N-(n-butyl) thiophosphoric triamide) on Earthworms (Eisenia fetida) reproduction according to the OECD Guidelines for Testing of Chemicals (1984) No 222 "Earthworm Reproduction Test" and EEC Commission Directive 87/302/EEC under GLP.

 

Eight solutions of NBPT and the deionized water (control) were mixed homogeneously into the soil which was filled in plastic containers before the earthworms were introduced on top of the soil at pH 5 -6. The test media was artificial soil comprised of approximately 70% sand, 20% Kaolin clay, and 10% finely ground sphagnum peat moss. The final moisture content in each test vessel was approximately 26-27%.

 

Forty worms per concentration (10 worms per replicate, 4 replicates per concentration) were exposed under continuous lighting to the following nominal concentrations of NBPT 0 (control), 87.79, 131.69, 197.53, 296.30, 444.44, 666.67, 1000 and 1500mg/kg dw at 19.2 -20.9°C. The earthworms were fed with cow manure spread on the soil surface of the containers once a week during the exposition period.

 

After 28 days of exposure the adult worms were removed from the soil and the number and weight of living adult worms were recorded to assess their mortality and their growth, respectively. During the exposure period, any clinical symptoms and the feed consumed were also recorded. After 56 days of the experiment (exposure+incubation periods), the juvenile worms hatched from cocoons were removed from the soil using water bath at a temperature 50-60oC and counted to assess the effect on reproduction. The observed mortalities or biomass decreases were not related to the concentration of NBPT and therefore no dose/effect values could be derived. No clinical symptoms were observed during the test and thefeed consumption was only decrease atthe highest tested concentration of1500 mg/kg soildw.

 

The number of juveniles was significantly decreased compared to the control group at the four highest test item concentrations of 444.44, 666.67, 1000 and 1500 mg/kg soil dry weight (Bonferroni t-test, p=0.05). The 56d-EC50for the reproduction rate (based on the juvenile numbers) ofEisenia fetidawas determined as 595.53 mg/kg soil dw based on a probit analysis. The 56d-NOAEC for the reproduction rate was estimated at 197.53 mg/kg soil dw.

Description of key information

There are three studies available for this end point, two acute and one chronic. The results are as follows:

The acute toxicity of Agrotain, to the earthworm Eisenia foetida, in an artificial soil test has been investigated. The 7 day LC50 for worms exposed to Agrotain is 860 mg a.i./kg (95% confidence interval = 600 to 1,000 mg a.i./kg), and the 14 day LC50 is 830 mg a.i./kg (95% confidence interval = 760 to 900 mg a.i./kg). The 7-day and 14-day LC50 values were equivalent to 1,100 mg a.i./kg when calculated on a dry weight basis. The NOEC is 600 mg a.i./kg (equivalent to 800 mg a.i./kg, calculated dry weight), based on survival, sublethal effects, time to burrow, and weight loss relative to the control.

In a 14-day toxicity study to earthworms (Eisenia fetida) with NBPT (N-(n-butyl) thiophosphoric triamide), the LC50 was determined as 1786.26 mg/kg soil dry weight (95 % confidence limits: 1628.16 – 2000.71 mg/kg soil dry weight). The NOEC related to mortality and biomass changes was 750 mg/kg soil dry weight.

The test item NBPT (N-(n-butyl) thiophosphoric triamide) doesn’t have an effect on the mortality or the biomass of the adult worms (after 28 days), but have an adverse effect on the reproduction of earthworms (Eisenia fetida) based on the number of juveniles observed after 56 days

For Eisenia fetida adult mortality and biomass development):

     LC50 (28 day): > 1500 mg Test Item/kg soil dry weight

     NOEC (28 day):     1500 mg/kg soil dry weight

      

For reproduction of Eisenia fedita based on the number of juveniles:

    EC50 (56 day):      595.53 mg/kg soil dry weight  (95 % confidence limits: 550.36 – 644.41 mg/kg soil dry weight)

    NOAEC (56 day):  197.53 mg Test Item/kg soil dry weight

    LOAEC (56 day):   296.30 mg Test Item /kg soil dry weight

As both acute studies are considerd reliable, the one with the 'worst case' result has been chosen as the key study.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
830 mg/kg soil dw
Long-term EC10, LC10 or NOEC for soil macroorganisms:
600 mg/kg soil dw

Additional information

A study was performed to assess the acute toxicity of the test material to the earthworm (Eisenia foetida) in an artificial soil. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 207 "Earthworm, Acute Toxicity Tests" and EEC Commission Directive 87/302/EEC.

The test substance, Agrotain [reported purity 25% active ingredient; N-(n-butyl) thiophosphoric triamide (NBPT)] was supplied by the sponsor.

The test was performed with five concentrations of test substance and a control at 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 substance was mixed with deionized water then added to dry artificial soil in each test vessel. The final moisture content in each test vessel was approximately 25%. All nominal calculations are based on milligrams of active ingredient per kilogram of wet soil, unless otherwise noted.

Forty worms per concentration (10 worms per replicate, 4 replicates per concentration) were exposed to the following nominal concentrations of Agrotain (control), 130,220,360, 600, and 1,000 mg a.i./kg. Exposure ofwonns to Agrot~ resulted in a 7 day median lethal concentration (LC50) of 860 mg a.i./kg (equivalent to 1,100 mg a.i./kg, calculated dry weight), and a 14 day LC50 of 830 mg a.i./kg (equivalent to 1,100 mg a.i./kg, calculated dry weight).

The acute toxicity of the test material, to the earthworm Eisenia foetida, in an artificial soil test has been investigated. The 7 day LC50 for worms exposed to Agrotain is 860 mg a.i./kg (95% confidence interval = 600 to 1,000 mg a.i./kg), and the 14 day LC50 is 830 mg a.i./kg (95% confidence interval = 760 to 900 mg a.i./kg). The 7-day and 14-day LC50 values were equivalent to 1,100 mg a.i./kg when calculated on a dry weight basis. The NOEC is 600 mg a.i./kg (equivalent to 800 mg a.i./kg, calculated dry weight), based on survival, sublethal effects, time to burrow, and weight loss relative to the control.