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Toxicity to soil microorganisms

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Reference
Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
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:
yes
Remarks:
minor deviations, no impact on outcome or the quality and integrity of the study
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
not required
Vehicle:
no
Details on preparation and application of test substrate:
The soil moisture content was determined.
Prior to final moistening at test start, the soil was adjusted to about 42% of its maximum water holding capacity with demineralised water. Drying out of the soil was prevented by moistening with demineralised water as necessary.

The soil was checked for a detectable microbial biomass (result expressed in terms of percentage of total organic carbon) and the amount of total inorganic nitrogen.

The soil amounts were amended with powdered lucerne-green-grass-meal (0.5% of soil dry weight). The C/N ratio was between 12/1 and 16/1. A ratio of 5 g Lucerne per kg of soil (dry weight was used.
Content of total inorganic nitrogen: 2.8% (dry weight)
Content of total organic carbon: 42.4% (dry weight)
C/N-ratio: 15.31
Particle size: 0.0288 - 1.008 mm
Origin: Alfalis, la maîtrise des luzernes, DESIALIS,
Société par actions simplifiée BP 124,
F-51007 Chalons en Champagne, France

Soil storage The soil was stored for 60 days (2014-07-18 to 2014-09-16) in the dark at 6 ± 2 °C in a climatic room (TE1200, VIESSMANN). Subsequently, the soil was pre-incubated at room temperature (ca.20 °C) for 21 days (2014-09-16 to 2014-10-07) to adapt the micro-organisms to test conditions before the start of the study.


3.2 Experimental Procedure

Test item Triethylenetetramine, TETA

Test concentrations 100 - 40 - 16 - 6.4 - 2.56 mg TETA/kg soil dry weight
(factor 2.5), equivalent to 38 - 15.2 - 6.1 - 2.4 - 0.97 mg N/kg soil dry weight, based on a calculated nitrogen content of 38%.

Reference item Cyanoguanidine is tested once every twelve months at concentrations of 50 and 100 mg/kg soil dry weight (laboratory ID: TBN1403, duration: 2014-04-02 to 2014-05-02). Results of the most recent test prior to this study are given in chapter 11.

Lot (Origin) / Expiry date STBC5050V (Sigma Aldrich) / 2015-02-18

Purity 99.5 %

CAS-RN 461-58-5

Control Untreated soil was tested under the same conditions as the test replicates.

Test duration 28 days

Replicates Triplicate vessels per treatment

Soil amount per replicate 400 g soil dry weight equivalent

Test vessels Plastic boxes (volume 1.0 L, food grade) with perforated tops to enable gas exchange. Incubation was performed in bulk and sub-samples were taken as stated below (see section 3.3).

Application The respective test item amounts were weighed out for each test item concentration and dissolved in demineralised water. Afterwards the test item solutions were mixed carefully into the soil with a mixer to ensure a homogeneous distribution of the test item in the soil. Subsequently the soil was distributed to the replicates. Exact amounts of demineralised water are listed in Table 1.

Frequency of application The application was carried out once at test initiation.

Table 1: Application Conditions of the Soil
Dry Weight (DW) before application [g/100 g soil] 86.9
Maximum water holding capacity (MWHC)* [g/100 g soil DW] 35.6 ± 1.7*
Soil moisture content in terms of percent MWHC before application [%] 42.4
Water amount to adjust to 45 % MWHC [g/100 g soil DW] 0.93
Water amount / test group [mL] 11.16
*) data provided by LUFA SPEYER

Temperature 20 ± 2 °C

Photoperiod Dark
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20 ± 2 °C
Moisture:
At test initiation the soil was adjusted to approximately 45% of its maximum water holding capacity. All replicates were weighed once per week to check for water losses by evaporation (recommended maximum 5%, actual 0.2 - 1.2%). demineralised water was added as necessary.
Details on test conditions:
Test item Triethylenetetramine, TETA

Test concentrations 100 - 40 - 16 - 6.4 - 2.56 mg TETA/kg soil dry weight
(factor 2.5), equivalent to 38 - 15.2 - 6.1 - 2.4 - 0.97 mg N/kg soil dry weight, based on a calculated nitrogen content of 38%.

Reference item Cyanoguanidine is tested once every twelve months at concentrations of 50 and 100 mg/kg soil dry weight (laboratory ID: TBN1403, duration: 2014-04-02 to 2014-05-02). Results of the most recent test prior to this study are given in chapter 11.

Lot (Origin) / Expiry date STBC5050V (Sigma Aldrich) / 2015-02-18

Purity 99.5 %

CAS-RN 461-58-5

Control Untreated soil was tested under the same conditions as the test replicates.

Test duration 28 days

Replicates Triplicate vessels per treatment

Soil amount per replicate 400 g soil dry weight equivalent

Test vessels Plastic boxes (volume 1.0 L, food grade) with perforated tops to enable gas exchange. Incubation was performed in bulk and sub-samples were taken as stated below (see section 3.3).

Application The respective test item amounts were weighed out for each test item concentration and dissolved in demineralised water. Afterwards the test item solutions were mixed carefully into the soil with a mixer to ensure a homogeneous distribution of the test item in the soil. Subsequently the soil was distributed to the replicates. Exact amounts of demineralised water are listed in Table 1.

Frequency of application The application was carried out once at test initiation.

Table 1: Application Conditions of the Soil
Dry Weight (DW) before application [g/100 g soil] 86.9
Maximum water holding capacity (MWHC)* [g/100 g soil DW] 35.6 ± 1.7*
Soil moisture content in terms of percent MWHC before application [%] 42.4
Water amount to adjust to 45 % MWHC [g/100 g soil DW] 0.93
Water amount / test group [mL] 11.16
*) data provided by LUFA SPEYER

Temperature 20 ± 2 °C

Photoperiod Dark


Soil Parameters
Parameter LUFA-soil 2.3
Batch-No. F2.32914
sampling depth* ca. 30 cm
pH value* 5.8 ± 0.7
Dry weight (DW) before application
[g/100 g soil] 86.9
Maximum water holding capacity*
[g/100 g DW] 35.6 ± 1.7
Particle size distribution acc. to DIN*
Sand:
2.0 - 0.63 mm % dry weight 2.5 ± 0.9
0.63 - 0.2 mm % dry weight 30.4 ± 0.6
0.2 - 0.063 mm % dry weight 24.7 ± 1.8
Silt:
0.063 - 0.02 mm % dry weight 19.5 ± 1.9
0.02 - 0.006 mm % dry weight 11.5 ± 0.7
0.006 - 0.002 mm % dry weight 5.0 ± 0.2
Clay:
< 0.002 mm % dry weight 6.4 ± 2.0
Organic carbon content %1) 0.68
Microbial biomass % of total organic carbon2) 2.90
Nitrate-N [mg NO3-N/kg DW]2) 8.88
Ammonium-N [mg NH4-N/kg DW]2) 0.435
Total inorganic Nitrogen [mg/kg DW]2) 9.32
Cation exchange capacity [meq / 100 g]* 7.3 ± 1.0
Soil texture* silty sand (uS)#
*) data provided by LUFA SPEYER
#) acc. to German DIN classification
1) determined by INSTITUT KOLDINGEN GMBH, based on dry weight (non GLP)
2) determined at day 2

Nominal and measured concentrations:
Nominal:
100 - 40 - 16 - 6.4 - 2.56 mg TETA/kg soil dry weight
(factor 2.5), equivalent to 38 - 15.2 - 6.1 - 2.4 - 0.97 mg N/kg soil dry weight, based on a calculated nitrogen content of 38%.
Reference substance (positive control):
yes
Remarks:
Cyanoguanidine (see above)
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Details on results:
Changes of nitrate-N contents and nitrate-N formation rates (expressed as percent of the control group values) are listed in Table 5 and Table 6.
The observed effects were qualitatively consistent with those of the preliminary range-finder test and concentrations of nitrate were higher than in the control at all treatments at every measurement point. There was no evidence of significant inhibition of the nitrate formation rate throughout the 28 day definitive test at all concentrations up to and including 100 mg TETA/kg soil dry weight, the highest concentration applied.

The cause of the dose-related increase in nitrate formation observed across the range of test item treatments is unknown. One explanation may be that TETA stimulated the breakdown of the lucerne meal substrate and accelerated the mineralisation of plant-derived organic nitrogen relative to the control. Alternatively (or additionally), partial breakdown of the test item may have released nitrogen for mineralisation; the nitrate-nitrogen concentrations in the TETA treatments at the end of the test exceeded the D 28 nitrate-nitrogen concentration in the control by amounts that are all within the range of the applied TETA treatments expressed as N equivalent concentrations. Discrimination between these two possibilities is not possible without making substantial alterations to the study design that are beyond the requirements and purpose of the test guideline.


Table 5: Reduction of Nitrate-N Content in soil containing TETA, relative to the untreated
control
Nitrate-N Content
Test concentration Inhibition [%] compared to control
[mg/kg soil dry weight] 0 d 7 d 14 d 28 d
2.56 -4 2 -4 0
6.4 0 -10 -3 -6
16 -4 -12 -6 -14
40 -12 -20 -12 -32
100 -16 -29 -34 -59
positive values = inhibition negative values = increase

Table 6: Inhibition of Nitrate-N Formation Rates in soil containing TETA, relative to the untreated
control
Nitrate-N Formation Rate
Test concentration Inhibition [%] compared to control
[mg/kg soil dry weight] 7 d 14 d 28 d
2.56 15 -5 2
6.4 -29* -6 -9
16 -27* -8 -19*
40 -34* -11 -43*
100 -55* -49* -83*
*) Statistically significant difference compared to control (ANOVA, p ≤ 0.05)
positive values = inhibition negative values = increase



Results with reference substance (positive control):
Reference Item Test

Cyanoguanidine a well-known inhibitor of nitrification is tested once per year as toxic reference with 50 and 100 mg/kg soil dry weight in LUFA soil of the same origin as used for studies with test items.

Table 13: Mean Nitrate-N Content in the Reference Item Test
Reference Item Concentration
[mg/kg soil dry weight] Mean ± SD of Nitrate-N Content [mg NO3-N/kg soil dry weight]
0 d 7 d 14 d 28 d
Control 16.1 ± 0.93 24.5 ± 0.46 33.6 ± 1.67 43.8 ± 1.66
50 16.0 ± 0.21 18.7 ± 1.67 18.3 ± 0.27 19.9 ± 0.65
100 17.8 ± 0.46 18.1 ± 0.21 18.5 ± 1.08 19.0 ± 0.96

Table 14: Inhibition of Nitrate-N Formation Rates of the Reference Item Test
Reference Item
Concentration Nitrate-N Formation Rate
[%] compared to Untreated Control
[mg/kg soil dry weight] 7 d 14 d 28 d
50 68* 87* 86*
100 97* 96* 96*
*) difference to control ≥ 25 %

The observed effects caused by the reference item Cyanoguanidine prove the sensitivity of the test system thereby showing its suitability for this kind of study.

Validity criteria fulfilled:
yes
Conclusions:
Conclusions

The effects of Triethylenetetramine, TETA on the the nitrohgen transformation activity of soil micro-organisms were determined according to OECD Guideline 216 (2000) with a dose-response test design in which the test item was applied at concentrations of 100, - 40, - 16, - 6.4 - and 2.56 mg/kg soil dry weight. Untreated LUFA 2.3 field soil, similarly mixed with a source of organic nitrogen (lucerne meal), was tested as the control under the same test conditions as the test item.

The effects of the test item on nitrogen transformation by soil microflora were determined by comparing rates of formation of the terminal transformation product, nitrate-nitrogen (NO3--N), in soil treated with TETA against the rate observed in the untreated control. Measurements of nitrate-nitogen concentrations in soil were made in all treatments and the control on the day of treatment application (day 0) and after 7, 14 and 28 days.

The rates of nitrate formation were consistently higher in all the TETA treatments than in the untreated control at all timepoints throughout the study. Consequently there was no evidence that exposure to TETA inhibited the nitrogen transformation acivity of soil microflora at the range of concentrations applied. The EC50 based on inhibition of nitrogen transformation processes could therefore not be determined reliably, but it exceeded 100 mg TETA/kg soil dry weight, the highest concentration applied in the definitive test. The corresponding no-observed effect concentration (NOEC) was 100 mg TETA/kg soil dw,

The cause of the dose-related increase in nitrate formation observed across the range of test item treatments is unknown. One explanation may be that TETA stimulated the breakdown of the lucerne meal substrate and accelerated the mineralisation of plant-derived organic nitrogen relative to the control. Alternatively (or additionally), partial breakdown of the test item may have released nitrogen for mineralisation; the nitrate-nitrogen concentrations in the TETA treatments at the end of the test exceeded the D 28 nitrate-nitrogen concentration in the control by amounts that are all within the range of the applied TETA treatments expressed as N equivalent concentrations. Discrimination between these two possibilities is not possible without making substantial alterations to the study design that are beyond the requirements and purpose of the test guideline.

Executive summary:

The effects of Triethylenetetramine, TETA (Batch no. P0968) on the metabolic activity of soil micro-organisms were determined according to OECD Guideline 216 (2000) at Dr.U.Noack-Laboratorien, D-31157 Sarstedt, Germany from 2014-10-07 to 2014-11-04. The test guideline presents alternative testing procedures that apply to agrochemical and non-agrochemical substances. The approach for non-agrochemical substances, which entails a dose-response test design and focuses primarily on inhibitory effects was adopted in this study performed with TETA.

The test item was applied via demineralised water at concentrations of 100, 40, 16, 6.4 and 2.56 mg/kg soil dry weight. Untreated field soil, adjusted to a moisture content corresponding to nominally 45% of its maximum water holding capacity and mixed with a source of organic nitrogen (lucerne meal), was tested as the control under the same test conditions as the test item replicates. Plastic boxes (volume 1.0 L) with perforated tops to enable gas exchange and filled with 400 g soil dry weight were used as test vessels.

In the presence of a viable community of soil microflora, organic nitrogen undergoes conversion to mineralised nitrogen and a sequence of microbially mediated transformations that result in the formation of a series of inorganic nitrogen species: ammonification(product = NH4+‑N), followed by Stage 1 nitrification (product = NO2-‑N) and finally Stage 2 nitrification (product = NO3--N). The effects of the test item on nitrogen transformation by soil microflora were determined by comparing rates of formation of the terminal transformation product, nitrate-nitrogen (NO3--N), in soil treated with TETA against the rate observed in the untreated control. Measurements of nitrate-nitrogen concentrations in soil were made in all treatments and the control on the day of treatment application (day 0) and after 7, 14 and 28 days.

 

The rates of nitrate formation were consistently higher in all the TETA treatments than in the untreated control at all timepoints throughout the study. Consequently there was no evidence that exposure to TETA inhibited the nitrogen transformation activity of soil microflora at the range of concentrations applied. The EC50based on inhibition of nitrogen transformation processes could therefore not be determined reliably, but it exceeded 100 mg TETA/kg soil dry weight, the highest concentration applied in the definitive test. The corresponding no-observed effect concentration (NOEC) was 100 mg TETA/kg soil dw. The cause of the dose-related increase in nitrate formation observed across the range of test item treatments is unknown. One explanation may be that TETA stimulated the breakdown of the lucerne meal substrate and accelerated the mineralisation of plant-derived organic nitrogen relative to the control. Alternatively (or additionally), partial breakdown of the test item may have released nitrogen for mineralisation; the nitrate-nitrogen concentrations in the TETA treatments at the end of the test exceeded the D 28 nitrate-nitrogen concentration in the control by amounts that are all within the range of the applied TETA treatments expressed as N‑equivalent concentrations. Discrimination between these two possibilities is not possible

Description of key information

NOEC 28 d):   100 mg/kg soil dw 

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:
100 mg/kg soil dw

Additional information

The effects of the substance on the metabolic activity of the nitrogen-N formation rate (nitrate) soil micro-organisms were determined according to OECD Guideline 216 over a 28 day period (GLP). The nitrogen transformation by soil microflora was determined by measuring the terminal transformation product, nitrate-nitrogen (NO3 –N). Following test substance concentrations were tested 2.56, 6.4, 16, 40 and 100 mg /kg soil dry weight. The nitrate-nitrogen concentrations were determined at the first day of application and at days 7, 14 and 28. The rates of nitrate formation were higher in all treatments compared to the untreated control at all time points. Thus an inhibition of the nitrogen transformation activity of soil microflora by the test substance was not demonstrated at the range of concentrations applied. The EC50 based on inhibition of nitrogen transformation processes could therefore not be determined, but it exceeded 100 mg test substance/kg soil dry weight, the highest concentration applied in the definitive test. The corresponding no-observed effect concentration (NOEC) was 100 mg/kg soil dw.