Phosphorus

Toxicity to soil microorganisms

Administrative data

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-04-26 to 2010-05-26

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

yes

Remarks:

Nitrate analysis was done on day 8,14,28 instead of day 7,14,28. The soil was stored at 6 +/- 2 °C instead of 4 +/- 2 °C due to organisational reasons.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Phosphorus

EC Number:

231-768-7

EC Name:

Phosphorus

Cas Number:

7723-14-0

IUPAC Name:

phosphine

Details on test material:

- Name of test material (as cited in study report): Red Phosphorus, untreated
- Molecular weight: Polymeric
- Physical state: Solid, dark reddish brown solid powder
- Analytical purity: 99.2 % (w/w)
- Purity test date: Not specified
- Lot/batch No.: Fra 07-23
- Expiration date of the lot/batch: 2011-04-24
- Stability under test conditions: Not specified
- Storage condition of test material: Room temperature, protected from moisture and light, under nitrogen gas to prevent oxidising.

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

No analytical monitoring

Test substrate

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. Bulk samples were prepared and divided into three replicates per test item concentration.



VEHICLE:
None

Test organisms

Test organisms (inoculum):

soil

Study design

Total exposure duration:

28 d

Test conditions

Test temperature:

nominal: 20 +/- 2 °C, actual: 20 - 21 °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:
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. F2.30310
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) 1.3
Microbial biomass (%) of total organic carbon 1.32
Nitrate-N [mg/kg DW] 7.52
Ammonium-N [mg/kg DW] < LOQ
Total inorganic Nitrogen [mg/kg DW] 7.52
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): 4.34
- 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 -14 -4 7
100 mg/kg soil dry weight -9 -8 -5
1000 mg/kg soil dry weight -66 -42 1

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

Results and discussion

Effect concentrations

Duration:

28 d

Dose descriptor:

other: NOEC (day 14 and 28)

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:

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

Results with reference substance (positive control):

Cyanoguanidin is tested once per year as toxic reference with 50 and 100 mg/kg DW.
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:

ECx values of the nitrate-N formation rate were not calculated since no dose-response effect was found.

Any other information on results incl. tables

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	-3	-12	2	-3
125	-6	-11	0	-5
250	3	-10	8	-8
500	-3	-6	-3	-9
1000	7	-2	1	2

Inhibition of Nitrate-N Formation Rates

Test concentration	[%] compared to Control
[mg/kg soil dry weight]	8 d	14 d	28 d
62.5	-71	13	-4
125	-45	13	-6
250	-95	21	-22
500	-26	-4	-17
1000	-57	-13	-4

Soil Dry Weights, Maximal Water Holding Capacity and pH-Values after 0 and 28 days

Test concentration	Soil Dry Weights [%]		Moisture content as [%] of the Maximal Water Holding Capacity		pH-Values
[mg/kg soil dry weight]	day 0	day 28	day 0	day 28	day 0	day 28
Control	84.0	84.4	54.1	52.7	7.02	6.43
62.5	84.0	84.2	54.2	53.5	7.11	6.58
125	84.2	84.1	53.6	54.0	7.12	6.57
250	84.0	84.3	54.3	53.0	7.11	6.58
500	83.9	84.4	54.5	52.8	7.09	6.47
1000	84.1	84.5	53.9	52.4	7.08	6.28

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

After 8 days of exposure, Red Phosphorus, untreated stimulated the nitrate formation rate throughout all test item concentrations compared to the control. This stimulation was abolished after 14 days of exposure with no evident (< 25 %) nitrate formation inhibition or stimulation at all test item concentrations. Accordingly, no inhibition or stimulation ≥ 25 % was observed after 28 days of exposure to Red Phosphorus, untreated at all test item concentrations.

The NOEC for nitrate transformation in soil on day 8 was determined to be < 62.5 mg/kg soil dry weight. The NOEC for nitrate transformation in soil on day 14 and 28 was set at 1000 mg/kg soil dry weight. Thus, Red Phosphorus, untreated is not expected to cause any long term detrimental effects on nitrogen turnover in soil under normal conditions.

Executive summary:

The effects of Red Phosphorus, untreated  (Batch no. Fra 07-23) on the metabolic activity of soil micro-organisms were determined according to OECD Guideline 216 (2000) at Dr.U.Noack-Laboratorien, 31157 , , from April 26^thto May 26^th, 2010. The test item was applied with the test item concentrations 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 52.4 - 54.5 % (measured) of the maximum water holding capacity and a pH-value between 6.28 and 7.12. 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, Red Phosphorus, untreated stimulated the nitrate formation rate throughout all test item concentrations from 26 to 95 % compared to the control. This stimulation was abolished after 14 days of exposure with no evident ( 25 %) nitrate formation inhibition or stimulation at all test item concentrations. Accordingly, no inhibition or stimulation ≥  25 % was observed after 28 days of exposure to Red Phosphorus, untreated at all test item concentrations.

Throughout the entire study inhibitions or stimulations of the nitrate transformation did not show a dose response effect. Presumably, the soil microflora is only directly affected at the particle surface but not at other soil parts. Anyhow, no evident effects of Red Phosphorus, untreated appeared after 14 and 28 days of exposure.  

The NOEC for nitrate transformation in soil on day 8 was determined to be 62.5 mg/kg soil dry weight. The NOEC for nitrate transformation in soil on day 14 and 28 was set at 1000 mg/kg soil dry weight. Thus, Red Phosphorus, untreated is not expected to cause any long term detrimental effects on nitrogen turnover in soil under normal conditions.