6-Amino-5-chloro-2-cyclopyrimidine-4-carboxylic acid

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Mar - 23 Apr Jun 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Version / remarks:

2000

Qualifier:

according to guideline

Guideline:

OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)

Version / remarks:

2000

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: For the nitrogen transformation the soil was thoroughly mixed with the ground lucerne meal before application. The final concentration of the dried lucerne meal was 0.5% of the soil dry weight.
For the short-term respiration the amount of glucose needed to obtain maximum shortterm rates of respiration in the test soil was determined prior to the beginning of the test. A concentration of 2 g glucose/kg soil wet weight was found to be the optimum and was used in the test.

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: 20 mg of the test substance was dissolved in 100 mL deionised water. Appropriate amounts of this stock solution were mixed into the soil. Deionised water was added to the soil to reach the desired range of soil moisture, resulting in a final water
content of 45 to 51% of the WHCmax.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20° ± 2°C

Moisture:

45 - 53% of the WHCmax

Organic carbon content (% dry weight):

1.16

Details on test conditions:

TEST SYSTEM
- Test container: 500 mL plastic boxes with perforated plastic lids (nitrogen transformation test) / 1000 mL plastic boxes, with perforated plastic lids (short-term respiration test)
- Amount of dry soil: 400 g (nitrogen transformation test) / 800 g (short-term respiration test)
- No. of replicates per concentration: 3
- No. of replicates per control: 3

SOIL INCUBATION
- Method: bulk

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site: longitude 8° 44' 38.70'' E, latitude 49° 51' 59.59'' N
- History of site: For at least four years prior to test initiation, no pesticides had been used on the soil. No organic or mineral fertilizer had been applied to the soils for two years prior to study initiation, respectively.
- Treatments with pesticides or fertilizers: no
- Accidental contamination: no
- Other: sampled (according ISO 10381-6, sampling depth 0.05 to 0.2 m)
- Soil texture
- % sand: 57.5
- % silt: 33.1
- % clay: 9.4
- Soil taxonomic classification: mid loamy sand
- pH (in water): 6.9
- Initial nitrate concentration for nitrogen transformation test: 16.425 mg/kg dry weight
- Maximum water holding capacity (in % dry weigth): 41.6
- Cation exchange capacity (mmol/kg): 46.25 mmol Ba/kg dry weight
- Pretreatment of soil: air dried and sieved (mesh 2 mm) at room temperature, preincubated at 46% of its maximum water hold capacity in ventilated boxes at about 20°C before used in the experiment
- Initial microbial biomass as % of total organic C: 1.55
- Storage (condition, duration): 3 weeks at 20° ± 2°C


EFFECT PARAMETERS MEASURED: nitrogen transformation and soil respiration (days 0, 7 (8), 14, and 28 days following application of the test item)

Nominal and measured concentrations:

Nominal test substance concentrations: 0.74 and 3.68 mg/kg dw

Reference substance (positive control):

yes

Remarks:

sodium chloride (tested at least once a year at the test facility)

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

>= 3.68 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks:

respiration rate

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

>= 3.68 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks:

respriration rate

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 3.68 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks:

respiration rate

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: Difference from control: soil respiration rates: -67.28%, nitrate-N content: -67.74, cumulative nitrate formation rate: -107.79%

Reported statistics and error estimates:

Data for the short-term respiration and the soil nitrate contents were tested for normality and homogeneity of variance using the R/S-Test Test (α = 0.05) and Cochran´s-Test ( = 0.05). Where the criterion of homogeneity was fulfilled, the Student t-test and in case of inhomogeneity the Welch t-test (pair-wise comparison, two sided, α = 0.05) was used for comparison of treated and control values.

Table 1: Summary of effects of Aminocyclopyrachlor on nitrate formation and short- term respiration in soil

 

Amino- cyclo- pyra- chlor concen- trationa	NO3–N levels (Day 28)		Nitrate formation rate (Day 0 to 28)		Nitrate formation rate (Day 14 to 28)		Respiration rate (Day 28)
	mg/kg dry soil	% Dev. from control bc	mg/kg dry soil/day	% Dev. from control bc	mg/kg dry soil/day	% Dev. from control bc	mg CO2/hr /kg dry soil	% Dev. from controlbc
Control	31.657	---	0.53	---	1.37	---	8.443	---
0.74 mg/kg sdw	29.784	-5.92	0.49	-7.55	1.29	-5.84	8.016*	-5.06
3.68 mg/kg sdw	28.489	-10.01	0.45	-15.09	1.26	-8.03	7.966*	-5.65

a     Test item concentrations correspond to 1- and 5-times the maximum PECsoil for Aminocyclopyrachlor

b    Negative value =% inhibition, positive value =% stimulation

c    Statistical evaluation (Student t-test + Welch t-test, two-sided, α = 0.05):

*significant differences from the control

Table 3: Variation of Replicate Control samples: Nitrogen and carbon transformation test: nitrate content

	CV1)%
day	nitrate content	soil respiration
0	0.46	2.67
72)	14.70	2.08
14	5.58	2.48
28	9.54	2.42

1)         Coefficient of variation

2)         Day 8 due to measurement failure on day 7 for soil respiration

Table 2: Validity criteria for OECD 216 and 217

Criterion from the guideline	Outcome	Validity criterion fulfilled
The variation between replicate control samples should be less than ± 15%.	0.46 - 14.7	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

NOEC (28 d) ≥ 3.68 mg/kg soil dw (nominal, OECD 216 & 217, nitrogen & carbon transformation)

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

3.68 mg/kg soil dw

Additional information

The effects of aminocyclopyrachlor on soil microflora were assessed in a test that measured nitrogen transformation and short-term respiration following an application of the test substance to soil. The test was conducted in accordance with the OECD 216 and 217. The test substance was incubated in a single mid loamy sand soil over a period of 28 days (carbon transformation) and 28 days (nitrogen transformation) at concentrations of 0.74 mg /kg soil dry weight and 3.68 mg/kg soil dry weight. Deviations from the control in nitrate levels, nitrate formation rates and rates of short-term respiration in soil treated with up to and including 3.68 mg/kg soil dry weight were low and all <25% indicating no significant effect occurred in carbon or nitrogen transformation. The derived EC10 and EC50 values were > 3.68 mg/kg soil dry weight.