Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to soil microorganisms

Currently viewing:

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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.21 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
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 concentrationsopen allclose all
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
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.