Lime (chemical), hydraulic

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 23, 2007 - August 28, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

BBA Part VI, 1-1

Deviations:

no

Principles of method if other than guideline:

Guideline: BBA VI, 1-1 (1990) under consideration of OECD 216 (2000) and OECD 217 (2000).

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Test organisms (inoculum):

soil

Total exposure duration:

96 d

Moisture:

Water content soil: 10.12 g/100 g soil d.w.
Water holding capacity: 39.70 g/100 g soil d.w.

Duration:

96 d

Dose descriptor:

NOEC

Effect conc.:

4 g/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dehydrogenase activity

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 12 g/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dehydrogenase activity

Duration:

48 d

Dose descriptor:

EC50

Effect conc.:

8.1 g/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dehydrogenase activity

Duration:

96 d

Dose descriptor:

EC50

Effect conc.:

8.7 g/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dehydrogenase activity

Validity criteria fulfilled:

yes

Remarks:

Reference item must have an effect of at least 15% on day 28 after treatment. In the most recent study the reference item Dinoterb caused an inhibition of dehydrogenase activity of -69.4, -73.4 and -84.4% at 6.80, 16.00 and 27.00 mg per kg soil d.w.

Conclusions:

The pH of the soil increased with increasing concentrations of calcium dihydroxide. The high pH value of the soil is considered to be the toxic effect. At the highest concentration tested, the maximum pH level was 11.9 which decreased to 8.5 during the course of the study. At low test item concentration the dehydrogenase activity was stimulated.

Reference 2

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

April 18, 2007 - July 25, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

SOIL
- on days 0 (3 hours), 14, 28, 48 and 96 after application, soil samples were taken for the determination of the mineral nitrogen content of the soil.

Vehicle:

yes

Details on preparation and application of test substrate:

COLLECTION
- soil removal: to a depth of 20 cm as mixed sample
- dried at room temperature
- passed through a 2 mm mesh sieve
- characterization: biologically active agricultural loamy sand soil (1.42% organic C, 9.9% clay, 39% silt, 51.2% sand), no fertilisation since 2003, last application of plant protection products in 1990
- origin: wassergut Canitz, Germany (12.694435960 degrees East, 51.403774567 degrees North)

STORAGE
- at a temperature of 4 °C
- in aerobic conditions in the dark
- before applicatin soil was adapted to test conditions

PREPARATION
1)
- 300 g soil (dry weight) (= one sub-sample) weighed per test vessel
- soil mixed with 0.5 % lucerne meal (i.e. 1.5 g/300 g soil d.w.) by means of a hand-stirrer
- vehicle: quartz meal
- water was added to the soil to achieve a water content of approx. 55 % of WHC
- incubation in wide-mouth glass flasks (500 mL) the below described test conditions
2)
- an additional soil sample (without lucerne meal) used for determination of initial NH4-N- and NO3-N-content
- NO3-N-content was 0.96 mg/100 g soil d.w.

Test organisms (inoculum):

soil

Total exposure duration:

96 d

Test temperature:

19.9 - 21.9°C in a climatic room

Moisture:

18.66-19.70 g/100 g soil d.w.

Details on test conditions:

- Illumination: darkness
- 3 replicates per concentrations (i.e. 3 subsamples of soil per concentration)
- humus content of soil: 2.44%
- initial pH 7.1
- microbial biomass 23.55 mg C/100 g soil d.w. (i.e. 1.66% compared to organic C content)

Nominal and measured concentrations:

nominal concentrations: 0, 1, 2, 4, 8, 10 and 12 g test item/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

Dinoterb

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

8 g/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: nitrogen transformation

Duration:

48 d

Dose descriptor:

NOEC

Effect conc.:

10 g/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: nitrogen transformation

Duration:

96 d

Dose descriptor:

NOEC

Effect conc.:

>= 12 g/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: nitrogen transformation

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

9.7 g/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: nitrogen transformation

Duration:

48 d

Dose descriptor:

EC50

Effect conc.:

> 10 g/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: nitrogen transformation

Duration:

96 d

Dose descriptor:

EC50

Effect conc.:

> 12 g/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: nitrogen transformation

Details on results:

not applicable

Results with reference substance (positive control):

Dinoterb caused a stimulation of nitrogen transformation of 44.8% and 58.2% at 6.80 and 16.00 mg Dinoterb per kg soil dry weight, respectively, 28 days after application.

Reported statistics and error estimates:

Not applicale

Validity criteria fulfilled:

yes

Remarks:

The coefficients of variation in the control group of the nitrogen transformation test were maximum 9.3% and thus fulfilled the demanded range (≤ 15%).

Conclusions:

The 96d-NOEC was 12 g/kg soil dry weight, the highest concentration tested. The pH of the soil increased with increasing concentrations of calcium dihydroxide. The high pH value of the soil is considered to be the toxic effect. At the highest concentration tested (12 g/kg soil), the maximum pH level was 11.9 which decreased to 8.5 during the course of the study. At high test concentrations, the nitrogen transformation activity of the soil microflora was shown to recover within 100 days of exposure to the test item. At low test item concentrations the nitrogen transformation was stimulated.

Reference 3

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

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)

Analytical monitoring:

no

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 (inoculum):

soil

Total exposure duration:

28 d

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

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.

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]

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.

Reference 4

Endpoint:

toxicity to soil microorganisms

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Common functional groups/mechanism of action.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Target: Lie (chemical), hydraulic [CAS 85117-09-5; See section 1.2 for information on purity.
Source: calcium dihydroxide [CAS 1305-62-0; EC 215-137-3] 98.2%

3. ANALOGUE APPROACH JUSTIFICATION
In the environment, lime substances rapidly dissociate or react with water. These reactions, together with the equivalent amount of hydroxyl ions set free when considering 100mg of the lime compound (hypothetic example), are illustrated below:
Ca(OH)2 <-> Ca2+ + 2OH-
100 mg Ca(OH)2 or 1.35 mmol sets free 2.70 mmol OH-
Ca(OH)2 + Ca2SiO4 + CaCO3 + 3 H2O <-> 4Ca2+ + SiO2 + CO2 + 8OH-
100 mg lime (chemical) hydraulic or 0.27 mmol sets free 2.16 mmol OH-
It has to be noted that CO32- is not expected to directly release two hydroxyl ions under most environmental conditions (depends on CO2 concentrations and pH) and this is therefore a worst case assumption.
From these reactions it is clear that the effect of lime (chemical) hydraulic will be caused either by calcium or hydroxyl ions. Since calcium is abundantly present in the environment and since the effect concentrations are within the same order of magnitude of its natural concentration, it can be assumed that the adverse effects are mainly caused by the pH increase caused by the hydroxyl ions. Furthermore, the above mentioned calculations show that the base equivalents are within a factor 2 for lime (chemical) hydraulic and calcium hydroxide. As such, it can be reasonably expected that the effect on pH of lime (chemical) hydraulic is comparable to calcium hydroxide for a same application on a weight basis. Consequently, read-across from calcium hydroxide to lime (chemical) hydraulic is justified.

4. DATA MATRIX
Source: No studies available
Target: 96d-EC50 = 8.7 g/kg soil dw. and 96d-NOEC = 4 g/kg soil dw. for calcium dihydroxide for dehydrogenase activity; 96d-EC50 > 12 g/kg soil dw. and 96d-NOEC >= 12 g/kg soil dw. for calcium dihydroxide for nitrogen transformation

Reason / purpose for cross-reference:

read-across source

Duration:

96 d

Dose descriptor:

NOEC

Effect conc.:

4 g/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dehydrogenase activity

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 12 g/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dehydrogenase activity

Duration:

48 d

Dose descriptor:

EC50

Effect conc.:

8.1 g/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dehydrogenase activity

Duration:

96 d

Dose descriptor:

EC50

Effect conc.:

8.7 g/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dehydrogenase activity

Description of key information

Klimisch 1 study: 96d-EC50 = 8.7 g/kg soil dw. and 96d-NOEC = 4 g/kg soil dw. for calcium dihydroxide for dehydrogenase activity (Schulz, 2007a)

Klimisch 1 study: 96d-EC50 > 12 g/kg soil dw. and 96d-NOEC >= 12 g/kg soil dw. for calcium dihydroxide for nitrogen transformation (Schulz, 2007b)

Klimisch 1 study: 28-d EC50 >1000 mg/kg soil dw and the 28-d NOEC = 1000 mg/kg soil dw.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

4 000 mg/kg soil dw

Additional information

The chronic study on the effect of calcium dihydroxide on the dehydrogenase activity in an agricultural loamy sand soil (Schulz, 2007a) was conducted according to German guidelines for testing of plant protection products (BBA VI, 1-1, 1990). The methods and results are well documented. As such a Klimisch 1 score was assigned to the study. The pH of the soil increased with increasing concentrations of calcium dihydroxide. The high pH value of the soil is considered to be the toxic effect. At the highest concentration tested, the maximum pH level was 11.9 which decreased to 8.5 during the course of the study. At low test item concentration the dehydrogenase activity was stimulated.

The chronic study on the effect of calcium dihydroxide on the nitrogen transformation in an agricultural loamy sand soil (Schulz, 2007b) was carried out according to OECD 216. The study is well documented, all validity criteria are fulfilled. As such a Klimisch 1 score was assigned to the study. The 96d-NOEC was 12 g/kg soil dry weight, the highest concentration tested. The pH of the soil increased with increasing concentrations of calcium dihydroxide. The high pH value of the soil is considered to be the toxic effect. At the highest concentration tested (12 g/kg soil), the maximum pH level was 11.9 which decreased to 8.5 during the course of the study. At high test concentrations, the nitrogen transformation activity of the soil microflora was shown to recover within 100 days of exposure to the test item. At low test item concentrations the nitrogen transformation was stimulated.

The chronic effects of calcium carbonate (nano) at a nominal concentration of 1000 mg/kg soil dw on the nitrogen transformation activity of soil microorganisms were assessed in a study performed to OECD TG 216 under GLP (Clarke, 2010). No adverse effects on the nitrogen transformation rate were exhibited at the concentration tested. Hence, the 28 day EC50 for calcium carbonate (nano) was found to be >1000 mg/kg soil dw and the NOEC was 1000mg/kg soil dw. Calcium carbonate is therefore not toxic to soil microorganisms.

In both studies performed with calcium dihydroxide, effects due to pH were seen at high concentrations. Over time, the pH dropped, presumably as the calcium was converted to calcium carbonate and its availability decreased. Therefore it may be concluded that calcium dihydroxide of high purity represents a worse-case for all grades of lime (chemical) hydraulic, although in any case toxicity to soil microorganisms is low.