[No public or meaningful name is available]

Toxicity to soil microorganisms

Administrative data

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

16 December 2009 to 17 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.21 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Calcium carbonate

EC Number:

207-439-9

EC Name:

Calcium carbonate

Cas Number:

471-34-1

Molecular formula:

CH2O3.Ca

IUPAC Name:

calcium carbonate

Test material form:

solid: nanoform, no surface treatment

Details on test material:

- Name of test material (as cited in study report): Calcium carbonate (nano)
- Description: White powder
- Batch No.: GICM014427
- Expiry date: 31 July 2014
- Storage: Room temperature in the dark

The nano form of calcium carbonate was tested because this form was anticipated to represent the worst case as it is likely to be more soluble than the bulk form due to the smaller particle size and hence greater surface area.

Specific details on test material used for the study:

Uncoated nano CaCO3

Sampling and analysis

Analytical monitoring:

no

Test substrate

Vehicle:

no

Details on preparation and application of test substrate:

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The test item was mixed with quartz sand prior to dispersal in soil. An amount of test item (1000 mg) was mixed with dry quartz sand (10 g). The test item/sand combination and an amount of powdered Lucerne-green-grass-meal (5 g) was then added to a final bulk soil weight of 1000 g (dry weight) and thoroughly mixed. An aliquot (47 mL) of water was then added to give the required test concentration of 1000 mg/kg with a nominal moisture content of 40% of the Water Holding Capacity (WHC). The soil was then mixed again to ensure homogeneity prior to splitting into 18 replicates for incubation.

Test organisms

Test organisms (inoculum):

soil

Study design

Total exposure duration:

28 d

Test conditions

Test temperature:

21 ± 1 °C

Moisture:

The moisture content of the soil, expressed as a percentage of the dry weight, was determined to be 9%. The Water Holding Capacity (WHC) of the soil as supplied was 35.1 g/100g and hence 47 mL of deionised reverse osmosis water per 1.0 kg of soil was added. This gave a final water content of 13.3 g/100 g i.e. 40% of the WHC as recommended by the Test Guidelines.

Details on test conditions:

TEST SYSTEM
- Test container (type, material, size): The soil samples were incubated in glass jars. The test vessels were covered with loosely fitted lids in order to minimise moisture loss by evaporation and maintained in a temperature controlled room at approximately 21 °C, in darkness.
- Amount of soil: 50 g (dry weight) of soil was used per vessel
- No. of replicates per concentration: 18 replicates
- No. of replicates per control: 9 replicates

SOIL INCUBATION
- Method: series of individual subsamples

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): LUFA Speyer, Obere Langgasse 40, 67346 Speyer, Germany
- Vegetation cover: Uncultivated
- Treatments with pesticides or fertilizers: The sampling site had not been treated with crop protection products or organic fertiliser for at least 3 years prior to sampling.
- Depth of sampling: 20 cm
- Soil texture
- % sand: 61.9 ± 4.2%
- Soil classification system: Sandy loam
- pH (in water): 6.6 ± 0.5
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 2.5 mg/100 g (ammonium <0.1 mg/100 g)
- Maximum water holding capacity (in % dry weight): 35.1%
- Pretreatment of soil: None
- Initial microbial biomass as % of total organic C: 121 µg C/g (equivalent to 1.2% of the total soil organic carbon content)

DETAILS OF PREINCUBATION OF SOIL: Not applicable

EFFECT PARAMETERS MEASURED: On days 0, 7 and 28 triplicate control and six replicate test item vessels were sacrificed for nitrate analysis.
The contents of the test vessel were transferred to 500 mL polyethylene bottles and an aliquot (250 mL) of potassium chloride (0.1 M) added. The mixture was then shaken (150 rpm, 60 minutes) prior to removal of the aqueous phase by filtration (0.45 µm).
To an aliquot (35 mL) of acid mixture was added 5 mL of the particle free extract followed by 5 mL of 2,6-dimethylphenol solution. The mixture was thoroughly mixed and allowed to stand for 10 minutes prior to determination of the absorbance at 324 nm.
A calibration curve was prepared by measuring absorbance values at 324 nm of standard solutions of potassium nitrate at the following concentrations: 1.0, 5.0, 10, 15, 20 and 25 mg NO3- N/L. The standard solutions were treated in the same manner as the test samples. Linear regression analysis of the standard curve data produced an equation for the best-fit line into which the test sample extract absorbance values were substituted to determine the nitrate nitrogen concentration.
The concentration of nitrate (mg/L) in the test sample extracts was obtained by multiplying the nitrate nitrogen concentration (mg/L) by a factor of 4.427.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: Nominal concentrations: 100 and 1000 mg/kg
- Results used to determine the conditions for the definitive study: The results showed no significant effect on nitrogen transformation activity.

Nominal and measured concentrations:

Nominal concentration: 1000 mg/kg

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Effect concentrations 2

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

Statistical analysis of the nitrate concentration values was carried out for the control and test item group using a Students t-test. The test item group was shown to have similar or not significantly different nitrate concentrations as the control group (P≥0.05). It was therefore considered that the test item exhibited no adverse effect on the nitrogen transformation rate.

Any other information on results incl. tables

Table 1: AbsorbanceReadingsand Nitrate Concentrations from the Definitive Test

Nominal Concentration (mg/kg soil)		Day 0		Day 7		Day 28
		Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)	Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)	Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)
Control	R1	0.097	32.00	0.160	54.15	0.556	193.50
	R2	0.144	48.55	0.147	49.60	0.512	178.05
	R3	0.146	49.25	0.178	60.50	0.432	149.90
	Mean	-	43.27	-	54.75	-	173.82
	CV%	-	23	-	10	-	13
1000	R1	0.178	60.50	0.228	78.10	0.528	183.65
	R2	0.171	58.05	0.225	77.05	0.440	152.70
	R3	0.175	59.45	0.230	78.80	0.539	187.55
	R4	0.184	62.60	0.222	76.00	0.488	169.60
	R5	0.184	62.60	0.206	70.35	0.527	183.30
	R6	0.204	69.65	0.246	84.40	0.521	181.20
	Mean	-	62.14	-	77.45	-	176.33
	CV%	-	7	-	6	-	7

* Corrected for blank absorbance value

R1-R6 = Replicates 1 to 6

CV = Coefficient of Variation

Table 2: Inhibition of Nitrogen Transformation Activity

Nominal Concentration (mg/kg soil)	Day 7		Day 28
	Nitrate Formation Rate (mg NO3/kg/d)	% Inhibition	Nitrate Formation Rate (mg NO3/kg/d)	% Inhibition
Control	1.64	-	4.66	-
1000	2.19	[34]	4.08	12

[Increase in nitrate concentration as compared to controls]

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The variation between replicate control nitrification rates was less than 15% and therefore satisfied the validation criterion given in the Test Guidelines.

Conclusions:

The effect of the calcium carbonate (nano) on the nitrogen transformation activity of soil microorganisms has been investigated over a 28-day period and gave an EC50 value of greater than 1000 mg/kg. Correspondingly the NOEC was 1000 mg/kg.