Flue dust, portland cement

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:

2010-06-08 to 2010-07-08

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

yes

Principles of method if other than guideline:

Nitrate analysis was done at day 8, 14 and 28 instead of at day 7, 14 and 28 due to not plausible results at day 7.
The soil was stored at 6 +/- 2 °C instead of 4 ± 2 °C due to organisational reasons.

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

No analytical monitoring

Vehicle:

yes

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: A silty sand soil (uS acc. to German DIN classification, field fresh sampled) and amended with powdered Lucerne-grass-green meal (0.5 % of dry weight) was used.



APPLICATION OF TEST SUBSTANCE TO SOIL
- Method:The test item amount for each concentration was weighed out and mixed thoroughly with a sub-sample of the soil (10 g per replicate). The treated soil sub-sample was given to the remaining soil, which was moistened with demineralised water to a moisture of nominally 55 % of the maximum water holding capacity. Subsequently, the soil was thoroughly mixed to ensure a homogeneous distribution of the test item in the soil. Bulk samples were prepared and divided into three replicates per test item concentration.



VEHICLE:
None

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

nominal: 20 +/- 2 °C, actual: 20 - 22 °C

Moisture:

Dry weight before application: 86.1 g/100 g soil

Details on test conditions:

TEST SYSTEM
- Testing facility: DR.U.NOACK-LABORATORIEN
- Test container (type, material, size): Plastic boxes (volume 0.5 L, food grade) with perforated tops.
- Amount of soil: 450 g soil DW
- No. of replicates per concentration: 3
- No. of replicates per vehicle control: 3


SOIL INCUBATION
- Method: bulk approach


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Offenbach, Im Bildgarten Nr. 570, Rheinland-Pfalz, Germany
- History of site:
Cultures:Cultures:
2010: uncultivated
2009: uncultivated
2008: uncultivated
2007: uncultivated
2006: uncultivated
Fertilisation:
No organic fertilisation
2009: none
2008: none
2007: none
2006: None
2005: None
- Vegetation cover: Not specified
- Treatments with pesticides or fertilizers: No crop protection products applied during sampling year and 4 former years
- Accidental contamination: No
- Depth of sampling: 20 cm
- Soil texture
Parameter LUFA-soil 2.3
Batch-No. F2.31210
sampling depth* ca. 20 cm
pH-value* 6.6 ± 0.5
Dry weight (DW) before application [g/100 g soil] 86.1
Maximum water holding capacity*
[g/100 g DW] 35.1 ± 2.0
Particle size distribution acc. to DIN*
Sand:
0.63 – 2.0 mm (% DW) 2.5 ± 0.7
0.2 – 0.63 mm (% DW) 27.6 ± 1.8
0.063 – 0.2 mm (% DW) 29.5 ± 3.6
Silt:
0.02 – 0.063mm (% DW) 18.1 ± 2.7
0.006 – 0.02 mm (% DW) 9.0 ± 0.6
0.002 – 0.006 mm (% DW) 4.3 ± 0.7
Clay:
< 0.002 mm (% DW) 8.7 ± 1.6
Organic carbon content %1) 1.5
Microbial biomass (%) of total organic carbon 4.84
Nitrate-N [mg/kg DW] 25.7
Ammonium-N [mg/kg DW] < LOQ
Total inorganic Nitrogen [mg/kg DW] 25.7
Cation exchange capacity [mVal/100 g]* 9.0 ± 3.0
Soil texture * silty sand (uS)

- Soil taxonomic classification: silty sand
- Soil classification system: acc. to German DIN classification
- pH (in water): 6.6 +/- 0.5
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 25.7
- Maximum water holding capacity (in % dry weigth): 35.1 +/- 2.0
- Cation exchange capacity: 9 +/- 3 mVal/100 g
- Pretreatment of soil: - The soil was manually cleared of large objects and then sieved to a particle size of 2 mm
- The maximal water holding capacity and the pH-value were
determined by LUFA SPEYER.
- The soil moisture content was determined.
- The soil was adjusted to 55 % of its maximal water holding
capacity with demineralised water.
- The soil was checked for a detectable microbial biomass
(result in terms of percentage of total organic carbon).

- Storage (condition, duration): The soil was stored for 59 days (2010-02-08 to 2010-04-08) in the dark at 6 +/- 2 °C in a climatic room
- Initial microbial biomass as % of total organic C: 1.32


DETAILS OF PREINCUBATION OF SOIL (if any): The soil was pre-incubated at room temperature (ca. 20 °C) for 18 days before experimental starting to guarantee a temperature adaptation of the micro-organisms.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Measurements of nitrate were carried out after 0, 8, 14 and 28 days. The pH-values and moisture contents were determined
on day 0 and 28.
Nitrate was extracted from the soil with a mineral salt solution.
For the elimination of coloured organic matter in the extraction solutions a cleaning step with solid phase extraction (SPE) cartridges was carried out according to ISO 14238. Thereafter, nitrate was determined photometrically.



VEHICLE CONTROL PERFORMED: No


RANGE-FINDING STUDY
Range finder (non-GLP): Inhibition of Nitrate-N Formation Rate Compared to the Control
Nominal test item concentration
[mg/kg soil dry weight] Day after application
7 14 28
10 mg/kg soil dry weight 9 14 3
100 mg/kg soil dry weight 32 0 18
1000 mg/kg soil dry weight -36 -15 21

Nominal and measured concentrations:

nominal test concentrations:1000 - 500 - 250 - 125 - 62.5 mg/kg soil dry weight (DW)

Reference substance (positive control):

yes

Remarks:

Cyanoguanidin

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

501 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: (< 62.5 - 918)

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

Inhibition of Nitrate-N Formation Rates in the Reference Item Test
[%] compared to Control
Day 8 14 28
Cyanoguanidine 50 mg/kg soil dry weight 83 131 41
Cyanoguanidine 100 mg/kg soil dry weight 168 143 93

Reported statistics and error estimates:

EC10 and EC25 values of day 14 and 28 data were calculated by a dose-sigmoidal response regression. Calculations of the confidence intervals for EC10 and EC25 were carried out using standard procedures.

Inhibition of Nitrate-N Contents

Test concentration	[%] compared to Control
[mg/kg soil dry weight]	0 d	8 d	14 d	28 d
62.5	0	2	-2	0
125	-1	1	-1	0
250	-9	-4	0	2
500	-7	0	1	-2
1000	0	0	6	13

Inhibition of Nitrate-N Formation Rates

Test concentration	[%] compared to Control
[mg/kg soil dry weight]	8 d	14 d	28 d
62.5	13	-9	0
125	13	0	0
250	19	29	17
500	35	27	3
1000	0	25	31

Nitrate-N Formation Rate: NOEC, LOEC and EC- Values with Confidence Intervals

	Day 8	Day 14	Day 28
	[mg/kg soil dry weight]
EC50	> 1000	> 1000	> 1000
95 % confidence interval	–	–	–
EC25	> 1000	348	883
95 % confidence interval	–	165 - > 1000	607 - > 1000
EC10	62.5	147	501
95 % confidence interval	-	95.7 - 231	62.5 - 918
NOEC	1000	125	500

Validity criteria fulfilled:

yes

Conclusions:

After 8 days of exposure to Flue Dust T no dose response related inhibition or stimulation ≥ 25 % of the microbial nitrate formation rate was observed. After 14 days of exposure the nitrate formation rate was inhibited by 29, 27 and 25 % at 250, 500 and 1000 mg/kg soil dry weight compared to the control. The inhibition of the nitrate formation after 14 days was abolished after further 14 days at 250 and 500 mg/kg soil dry weight. However, at 1000 mg/kg soil dry weight an inhibition of 31 % was determined after 28 days of exposure.
Flue Dust T strongly increased the soil pH on day 0 which may be part of the inhibitory effects of Flue Dust T on the microbial nitrate formation rate. However, the pH difference was reduced after 28 days.

The NOEC for microbial nitrate transformation in soil on day 8 was determined to be at 1000 mg/kg soil dry weight. The NOEC at day 14 was set at 125 mg/kg soil dry weight with a calculated EC25 at 348 mg/kg soil dry weight. The NOEC for microbial nitrate transformation on day 28 was determined to be at 500 mg/kg soil dry weight with a calculated EC25 at 883 mg/kg soil dry weight.

Since, below and at 500 mg test item/kg soil dry weight, no inhibition or stimulation of nitrate formation ≥ 25 % was observed after 28 days of exposure, Flue Dust T is not expected to cause any long term detrimental effects at 500 mg/kg soil dry weight on nitrate-N formation rates under normal conditions.

Executive summary:

The effects of Flue Dust T (Batch no. 12-2009) on the metabolic activity of soil micro-organisms were determined according to OECD Guideline 216 (2000) at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany , from June 8^th to July 8^th, 2010. The test item was applied with the test item concentrations of 62.5 - 125 - 250 - 500 - 1000 mg/kg soil dry weight. Untreated standard silty sand soil was tested as control under the same test conditions as the test item concentrations. Plastic boxes (volume 1.0 L) with perforated tops to enable gas exchange and filled with 450 g soil dry weight, moistened to nominally 55 % of its maximal water holding capacity, were used.

The treated soils were incubated for 28 days at 51.1 and 52.4 % of the maximum water holding capacity and pH values between 7.10 and 8.24 measured at day 0. After 28 days of exposure the soil moisture ranged between 51.0 and 53.2 % of the maximum water holding capacity and the soil pH ranged between 7.17 and 7.60. The effects of the test item on nitrate transformation were determined on the day of treatment (day 0) and after 8, 14 and 28 days. The values were compared to the untreated control.

After 8 days of exposure to Flue Dust T no dose response related inhibition or stimulation ≥  25 % of the microbial nitrate formation rate was observed. After 14 days of exposure the nitrate formation rate was inhibited by 29, 27 and 25 % at 250, 500 and 1000 mg/kg soil dry weight compared to the control. The inhibition of the nitrate formation after 14 days was abolished after further 14 days at 250 and 500 mg/kg soil dry weight. However, at 1000 mg/kg soil dry weight an inhibition of 31 % was determined after 28 days of exposure (Figure 1).

It should be taken into account that Flue Dust T strongly increased the soil pH on day 0 which may be part of the inhibitory effects of Flue Dust T on the microbial nitrate formation rate. However, the pH difference was reduced after 28 days. 

The NOEC for microbial nitrate transformation in soil on day 8 was determined to be at 1000 mg/kg soil dry weight. The NOEC at day 14 was set at 125 mg/kg soil dry weight with a calculated EC₁₀ at 147 mg/kg soil dry weight and an EC₂₅at 348 mg/kg soil dry weight. The NOEC for microbial nitrate transformation on day 28 was determined to be at 500 mg/kg soil dry weight with an EC₁₀at 501 mg/kg soil dry weight and an EC₂₅ at 883 mg/kg soil dry weight (Table 1). Since, below and at 500 mg test item/kg soil dry weight, no inhibition or stimulation of nitrate formation ≥ 25 % was observed after 28 days of exposure, Flue Dust T is not expected to cause any long term detrimental effects at 500 mg/kg soil dry weight on nitrate-N formation rates under normal conditions.

Nitrate-N Formation Rate: NOEC, LOEC and EC- Values with Confidence Intervals

	Day 8	Day 14	Day 28
	[mg/kg soil dry weight]
EC50	> 1000	> 1000	> 1000
95 % confidence interval	–	–	–
EC25	> 1000	348	883
95 % confidence interval	–	165 - > 1000	607 - > 1000
EC10	62.5	147	501
95 % confidence interval	-	95.7 - 231	62.5 - 918
NOEC	1000	125	500

Description of key information

Due to a Klimesch 1 study, Flue Dust is not toxic to soil micro-organism (microbial nitrate transformation test, OECD TG 216) up to a concentration of 500 mg/kg soil dry weight.

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:

1 000 mg/kg soil dw

Long-term EC10 or NOEC for soil microorganisms:

500 mg/kg soil dw

Additional information

It has to taken into account that Flue dust strongly increased the soil pH, which may be part of the inhibitory effect, which was observed at higher concentrations. The pH increase comes from the presence of calcium oxide, which reacts with water to calcium hydroxide. Additional calcium hydroxide is formed by the hydration reaction of clinker phases, which takes place in presence of soil moisture. The other flue dust constituents are inorganic minerals, like calcium carbonate, quartz, potassium chloride, calcium sulfate, which are naturally occuring substances. These constituents should not have toxic effects on soil micro-organisms.