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Toxicity to soil microorganisms

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Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study is performed according to OECD 216 (2000) guideline and is well documented.
Qualifier:
according to
Guideline:
other: OECD 216 (2000): "Soil Microorganisms: Nitrogen Transformation Test”
Deviations:
no
GLP compliance:
yes
Remarks:
The study has no formal GLP certificate. However, the study was conducted at a GLP-certified laboratory, staff was appropriately trained and standard operating procedures were followed in terms of experimental performance and raw data documentation.
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
- The test item was dissolved in a sufficient amount of deionised water to prepare a stock solution. This stock solution was diluted with deionised water to obtain one test item solution (= dosage solution) for each test item concentration.
- Method of mixing into soil (if used): The test item was mixed into the soil of the respective treatment.
- soil was ammended with Lucerne-green-meal pellets. Nitrogen content (%): 2.79; Carbon content (%): 41.03; C/N ratio: 14.7
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20 +- 2 °C
Moisture:
40-60% WHCmax
Details on test conditions:
TEST SYSTEM
- Test container (material, size):Glass jars with lids (Model Sturz, volume 0.25 L) were used as test vessels.
- Amount of soil or substrate: 30 g fresh weight per test vessel
- No. of replicates per treatment group: 3
- No. of replicates per control: 3

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- The test system was a freshly collected and sieved field soil (Lufa F2.3) harbouring its natural microbial community.
- Sampling location: Germany, Rheinland-Pfalz, Offenbach, Im Bildgarten No. 507
- Sampling conditions: Depth: ca. 20 cm; Date: January 28, 2008
- Preparation: Air drying (only until the soil was sievable) from January 28 to February 11, 2008; final sieving to 2 mm on February 11, 2008.
- Storage: stored at ambient temperature in the dark until use.
- Organic carbon [%]: 0.99
- pH (0.01 M CaCl2): 6.2
- Cation exchange capacity [mval/100 g]: 7
- Max. water holding capacity [g/100 g]: 34.2
- Particle size according to USDA [mm]: Relative distribution [%]
< 0.002 9.3
0.002 - 0.05 29.1
0.05 - 2.0 61.6
- Soil type: sandy loam
- Soil moisture [% dry mass]: 7.1
- from 2006 till 2008: soil uncultivated. From 2004 till 2005: soil cultivated (pumpkin) and fertilization used (400kg/ha in 2004 and 300kg/ha in 2005). Fertilizer was Nirophoska Spezial (12% N, 12% P2O5, 17% K2O)

- soil was ammended with Lucerne-green-meal pellets. Nitrogen content (%): 2.79; Carbon content (%): 41.03; C/N ratio: 14.7

OTHER TEST CONDITIONS
- Following test item application the test vessels were incubated for 28 days at 20 ± 2 °C in the dark. All test vessels were weighted once a week and weight loss was compensated with deionised water to keep the soil moisture within a range of ± 5%.
Aqueous extracts were prepared by shaking 20 g dry mass equivalent of soil with 100 mL of a 0.01 M CaCl2 solution in a polyethylene flask at 150 rpm for 60 min followed by filtration through folded filters. Nitrate was measured photometrically.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :The concentration of nitrate was determined in aqueous extracts from soil aliquots on day 0 and day 28 after test item application.
Nominal and measured concentrations:
nominal added boric acid concentrations: control, 150, 300, 600, 1200 and 2400 mg/kg soil dry weight
i.e. 0, 26.2, 52.4, 104.9, 210 and 420 mg B/kg dw
Reference substance (positive control):
no
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
419.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
added B
Basis for effect:
nitrate formation rate
Details on results:
Control (C): The initial mean nitrate concentration of the bulk soil on day 0 of the test and prior to adding luzerne meal was 56.7 mg/kg soil. It increased to 127 mg/kg soil on day 28. The coefficient of variance of the control was 0.67% on day 0 and 6.25 on day 28.

A significantly increased NO3 concentration compared to the control was observed at boric acid concentrations of 300 mg/kg soil (15%), 600 mg/kg soil (59%) and 1200 mg/kg soil (30%). At 150 mg/kg soil and 2400 mg/kg soil NO3 concentrations in soil were not significantly different from the control. Since no adverse effect on the formation of NO3 in soil was observed, the NOEC was calculated to be >2400 mg/kg soil dry mass, i.e. >419.6 mg B/kg soil dry mass.
Reported statistics and error estimates:
Normal distribution and homogeneity of variance was assessed using the RS-test and the Cochran test, respectively. To detect differences between the untreated control and the test item treatments Dunnetts test procedure for homogeneous variances was applied.

28-d NOEC for nitrate formation rate: >= 2400 mg H3BO3/kg dry soil

Validity criteria fulfilled:
yes
Conclusions:
Study is performed according to OECD 216 (2000) guideline and is well documented. A reliable 28-d EC10 value for N transformation in soil of 419.6 mg B/ kg dry soil is obtained.
Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The value for nitrogen transformation is considered reliable and relevant for the effects asseement. The nitrification test fulfilled the validity criteria in the OECD216 Guideline.
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
Soil moisture during the test was adjusted to ca 50% of WHC (i.e. water content of 21.1% for the sandy loam and 17.8% for the humic acid soil).
Soils were amended with powdered lucerne meal (C/N ratio 13/1) at a ratio of 5 g/kg dw and thoroughly mixed.
A stock solution was prepared by dissolving 4.2618g of the test substance in 100 ml ultrapure water. from this stock solution dilutions were made in ultrapure water. Subsequently aliquots wer added to the soil samples that were mixed thoroughly through the soil.
Test organisms (inoculum):
soil
Total exposure duration:
102 d
Test temperature:
20 +/- 2°C
Moisture:
ca 50% of WHC (i.e. water content of 21.1% for the sandy loam and 17.8% for the humic acid soil)
Details on test conditions:
TEST SYSTEM
SOIL INCUBATION
- Method: bulk / series of individual subsamples: For each concentration and soil type, 1 kg ww was placed in a suitable container, amended with boric acid and incubated in the dark. Containers were covered with punctured glass covers to allow gas exchange, but to minimize the loss of moisture.

EXTRACTION
- Test container (type, material, size): not reported
- Amount of soil for each concentration and soil type: 25 g dry soil
- No. of replicates per concentration: 4

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)

soil A: sandy loam, taken from grassland located at the Maasdijk, Heerewaarden (50°48'N, 5 22' E) on 31 Jan 2001
microbial biomass: 128mg C/kg dw
- % sand: 63.4
- % silt: 24.0
- % clay: 12.6
- pH (1:5) in water 8.3
- pH (1:5) in 0.01 M CaCl2 7.6
- CEC (buffered at pH 8.1) 9.0 cmolc/kg
- microbial biomass: 128 mg C/kg dw

soil B: humic sand, taken at the Droevendaal experimental station, Wageningen (51°59'N, 5 39' E) on 31 Jan 2001
microbial biomass: 106mg C/kg dw
- % sand: 85.3
- % silt: 10.2
- % clay: 4.5
- pH (1:5) in water 6.0
- pH (1:5) in 0.01 M CaCl2 5.4
- CEC (buffered at pH 8.1) 12.9 cmolc/kg
- microbial biomass: 106 mg C/kg dw


Pretreatment of soil: before the start of the experiment the soil samples were partly air-dried until they could be sieved to obtain soil particles <= 2mm. The sieved soil samples were analysed. The moisture content of the soils was adjusted to approximately 50% of maximum water holding capacity (i.e. water content of 21.1% for the sandy loam and 17.8% for the humic acid soil). Soil samples were stored at 20 +/- 2°C for approximately one month to restore microbial activity.

nitrate concentrations were measured in a 0.1M KCl extract (1:5 soil/solution ratio) by ion chromatography (4 replicates per treatment)

Nominal and measured concentrations:
boric acid concentration series: 0.3, 3, 10, 30 and 100mg boric acid/kg dw
i.e. 0, 0.05, 0.52, 1.75, 5.25, 17.5 mg B/kg dw
Reference substance (positive control):
not specified
Duration:
102 d
Dose descriptor:
EC10
Effect conc.:
15.4 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
B
Basis for effect:
nitrate formation rate
Remarks on result:
other: in humic sand soil
Duration:
102 d
Dose descriptor:
EC50
Effect conc.:
> 17.5
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
B
Basis for effect:
nitrate formation rate
Remarks on result:
other: in humic sand soil
Duration:
102 d
Dose descriptor:
EC10
Effect conc.:
17.2 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
B
Basis for effect:
nitrate formation rate
Remarks on result:
other: sandy loam
Duration:
102 d
Dose descriptor:
EC50
Effect conc.:
> 17.5 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
B
Basis for effect:
nitrate formation rate
Remarks on result:
other: sandy loam
Details on results:
In the original study report, nitrification rates were calculated based on the soil nitrate concentration after x days, without taking the nitrate concentration at day 0 into account. Moreover a 25% effect level was used as treshold for significance. Therefore data analysis was repeated, based on the raw data reported. The nitrification rate was calculated: (nitrate concentration in the soil sample after x days - the nitrate concentration at day 0) / number of days)
However, this way only an average response (nitrification rate) could be calulated per treatment and no NOEC values could be derived.
EC10 values for nitrification rate were based on a log-logistic dose-response curve fitted by minimising unweighted squared residuals sum.
Validity criteria fulfilled:
not specified
Conclusions:
The value for nitrogen transformation is considered reliable and relevant for the effects assessment. The nitrification test fulfilled the validity criteria in the OECD216 Guideline.
Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: meets generally accepted scientific standards, well documented and acceptable for hazard assessment
Qualifier:
according to
Guideline:
ISO 14238
Deviations:
no
GLP compliance:
yes
Remarks:
The study has no formal GLP status, but it study was performed by qualified staff according to international guidelines and can therefore be considered as GLP-confom.
Analytical monitoring:
yes
Vehicle:
no
Details on preparation and application of test substrate:
A stock solution of the weak acid H3BO3 was made (25 g/l). Soils were spiked in doses within the range of 0-293 mg B/kg by mixing the B solutions in the soil using a laboratory spoon. Soils were tested both immediatedly after spiking and after 5 months equilibration (ageing).
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Remarks:
The total Substrate Induced Nitrification (SIN) was calculated 28 days after the amendment of the (NH4)2SO4 solution in freshly spiked and aged (5months) soils.
Test temperature:
20°C
Moisture:
75 % of pF 2
Details on test conditions:
TEST SYSTEM
- No. of replicates per concentration: 3

SOURCE AND PROPERTIES OF SUBSTRATE (if soil):
- Soil texture (if natural soil) E2
- % sand:81
- % silt: 8
- % clay: 2
- Soil texture (if natural soil) E4
- % sand: 8
- % silt: 70
- % clay: 12
- Soil texture (if natural soil) E6
- % sand: 20
- % silt: 50
- % clay: 10
- CEC values for reference soils (cmol/kg):
- Soil E2: 4.1
- Soil E4: 12.2
- Soil E6: 14.3
- pH (CaCl2)
- Soil E2: 5.2
- Soil E4: 6.7
- Soil E6: 7.3
- background B (mg/kg)
- Soil E2: 2.3
- Soil E4: 11
- Soil E6: 7.8

SOIL PREINCUBATION
- Method: Soil samples were pre-incubated at a moisture content equivalent to 50% of pF 2 for 7 days at 20°C. Uncontaminated soils were then spiked with H3BO3 to 7 concentrations (control plus six treatments). Additional deionized water was added together with the spike solution to adjust the soil moisture content to 75% of pF 2. All soils were thoroughly mixed after amendments using laboratory spoons. Moisture content in control soils was raised to 75% of pF 2 with deionized water only. Thereafter soils were amended with 100 mg NH4-N/kg fresh soil using a stock solution containing 80 mg (NH4)2SO4/ml.

EFFECT PARAMETERS MEASURED
The effect parameter measured is the substrate induced nitrification (SIN), which is the nitrification at limited substrate (NH4+) availability. Soil nitrate is measure colorimetrically in a centrifuged soil extract (1M KCl, 2.5 g subsample, L/S 4, 2h end-over-end shaking)

Nominal and measured concentrations:
nominal added concentrations: control, 3.0, 7.5, 18.8, 46.9, 117.2, 293.0 mg added B/kg dry matter
Reference substance (positive control):
no
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
12 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
added B
Basis for effect:
nitrate formation rate
Remarks on result:
other: for freshly spiked soil E2; 95% CI: 10-13 mg B/kg dw
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
3 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
added B
Basis for effect:
nitrate formation rate
Remarks on result:
other: for 5 months aged soil E2; 95% CI: 2-6 mg B/kg dw
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
72 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
added B
Basis for effect:
nitrate formation rate
Remarks on result:
other: for freshly spiked soil E4; 95% CI: 39-135 mg B/kg dw
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
61 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
added B
Basis for effect:
nitrate formation rate
Remarks on result:
other: for 5 months aged soil E4; 95% CI: 54-70 mg B/kg dw
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
226 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
added B
Basis for effect:
nitrate formation rate
Remarks on result:
other: for freshly spiked soil E6; 95% CI: 136-376 mg B/kg dw
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
139 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
added B
Basis for effect:
nitrate formation rate
Remarks on result:
other: for 5 months aged soil E6; 95% CI: 17-1116 mg B/kg dw
Details on results:
In soils E4 and E6 all ammonia was already nitrified in the control soils and the soils with the lowest B doses. Nitrification in these soils seems not to be very sensitive to B stress. Soil E2, however, shows already an effect of B at low doses. The lower pH and the sandy character of the soil involve a lower nitrification rate (not all ammonia is nitrified yet) and a low sorption capacity, which makes B more bioavailable and concomitantly more toxic. Ageing during 5 months did not increase ED10 values. In contrast, the ED10 values significantly decreased in soil E2
Reported statistics and error estimates:
Dose-response curves were fitted by a log-logistic model using the Marquardt Method.
The ED10/ED50 model was adjusted to account for hormesis effects when the root growth in the soils with added B was at least 20% higher compared to the control soil. The ‘dose’ in this model is the added B concentration (nominal concentration), and ED10 values and their 95% confidence interval, expressed as added B, are predicted from the appropriate parameters of the curve.

soil code age  EC50  95% confidence interval 
E2  freshly spiked  24  22 -26 
E2  aged  21  17 -27 
E4  freshly spiked  97  77 -124 
E4  aged  114  110 -118 
E6  freshly spiked  274  240 -312 
E6  aged  248  147 -421 

28 -d EC50 values for nitrogen transformation expressed as mg added B/kg dry soil

Validity criteria fulfilled:
yes
Conclusions:
Reliable study. EC10 values are used for PNEC derivation

Description of key information

Key value for chemical safety assessment

Additional information

In total 9 individual high quality NOEC/EC10 values (for 2 microbial processes) are selected for the PNEC derivation, ranging from 3 mg B/kg for substrate induced nitrification in Zwijnaarde soil (Van Laer et al., 2010) to 419.6 mg B/kg for nitrogen transformation (Förster and Becker, 2009b).