Registration Dossier

Administrative data

Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2015-07-01 to 2015-07- 30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP conditions with chemical analysis

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
no
Principles of method if other than guideline:
No
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Solution in water
Details on test material:
Chemical name: GLDA-Na4 aq. sol
Chemical name: L-glutamic acid N,N-diacetic acid tetrasodium salt
CAS No.: 051981-21-6
Batch Number: FC-C 11836
Active ingredient content: 47.5%
Appearance: Light yellow, clear liquid (max. 250 APHA)
Water solubility: Entirely soluble

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
All concentration levels and the control were analytically verified via LC-MS/MS at the test start (day 0) and test end (day 28) with external standard calibration. At test start, samples of the application solutions were taken directly. After 28 days, mixed soil samples out of the replicates were taken (overall amount of 10 – 20 g per treatment).
Nitrate was extracted from soil with a mineral salt solution. A cleaning step with solid phase extraction (SPE) cartridges was carried out to eliminate potential interference from coloured organic matter in the extraction solution. Nitrate-nitrogen concentrations were then determined photometrically
Wave-length 588 nm for nitrate
Solutions Extraction solution: Potassium chloride, 1 M and 2 M
Reagent: 2 g/L 4-Ethylresorcinol in 2-Propanol

Standard Potassium nitrate > 99%

Preparation of standard A stock solution of 100 mg Potassium nitrate/L was prepared in solutions demineralised water. 6 concentrations were prepared by dilution with 1 M potassium chloride and used for calibration.
Calibration Calibration was performed at experimental starting.
Working steps 15 g soil of each replicate were weighed into shaking flasks and shaken with 60 mL extraction solution for 1 h at 150 - 200 rpm. Mixtures were then filtered. The first 20 mL of each filtrate was rejected and the remainder collected for SPE clean-up and photometric analysis.

Sample clean-up 10 mL of each filtered extract was cleaned via C18-SPE-cartridges to remove dissolved and coloured organic matter which could interfere with the photometric determination. Conditioning of the cartridges was done with 2 x 2.5 mL methanol and thereafter with 2 x 2.5 mL demineralised water. Care was taken not to allow the cartridges to dry out. After conditioning, the sample was applied. The first 2.5 mL of each cleaned extract were rejected. The following volumes were stored in reagent tubes.

Nitrate determination 1 mL of the cleaned extract was diluted with 1 mL demineralised water. A cuvette was filled with 1.8 mL sulphuric acid (86 %). 0.5 mL of diluted sample was added. The cuvette was closed and shaken gently. After 15 min 0.3 mL of the reagent solution was added. After 45 min photometric determination at 588 nm was carried out. Extraction solution was used as ground signal. On day 0 all samples were diluted 1:2 with demineralised water (1 part sample and 2 parts dilution). On day 7 all samples, except the 1.replicate of 12.9 mg/kg DW and the 2. and 3.replicate of 53.1 mg/kg DW, were diluted 1:2. The other replicates were diluted 1:4 with 1 M potassium chloride. On day 14 all samples were diluted 1:4 before measurement. On day 28 samples were diluted 1:8 for the control and all test item concentrations before measurements.

Method validation
The linearity and limit of quantification (LOQ) of the nitrate method were evaluated. In the range of calibration standards the second lowest standard was chosen as the limit of quantification.

Test substrate

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 moisture 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: 2.92% (dry weight)
Content of total organic carbon: 43.6% (dry weight)
C/N-ratio: 14.9
Origin: HEUCOBS,
Burgstraße 9,
16259 Falkenberg / M.; Deutschland

The soil parameters determined at experimental starting at latest are listed below.

Soil Parameters
Parameter LUFA-soil 2.3
Batch-No. F2.31615
sampling depth* ca. 20 cm
pH value* 5.7 ± 0.6
Dry weight (DW) before application2)
[g/100 g soil] 87.3
Maximum water holding capacity*
[g/100 g DW] 35.4 ± 1.5
Particle size distribution acc. to DIN*
Sand:
2.0 - 0.63 mm % dry weight 2.7 ± 0.8
0.63 - 0.2 mm % dry weight 30.3 ± 0.6
0.2 - 0.063 mm % dry weight 24.6 ± 1.5
Silt:
0.063 - 0.02 mm % dry weight 19.0 ± 1.8
0.02 - 0.006 mm % dry weight 11.5 ± 0.6
0.006 - 0.002 mm % dry weight 5.4 ± 0.8
Clay:
< 0.002 mm % dry weight 6.6 ± 1.7
Organic carbon content %1) 0.56
Microbial biomass % of total organic carbon2) 4.09
Nitrate-N2) [mg NO3-N/kg DW] 12.7
Ammonium-N2) [mg NH4-N/kg DW] 0.53
Total inorganic Nitrogen [mg/kg DW] 13.2
Cation exchange capacity [meq / 100 g]* 7.5 ± 0.9
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 by DR.U.NOACK-LABORATORIEN



Soil site Offenbach, “rechts der Landauer Str.“ Nr. 826/7, Rheinland-Pfalz, Germany

Gauß-Krüger-Coordinates R-439683; H-5449554

Sampling date 2015-04-16

Soil history Cultures:
2011 - 2014: uncultivated

Fertilisation:
2015: none
2014: 3500 kg/ha CaO (05.06.2014 and 24.09.2014)
3500 kg/ha CaO; 1463 kg/ha MgO (15.12.2014)
2013: none
2012: none
2011: none

Pesticides No crop protection products applied during the year of sampling and 4 preceding years.

Test organisms

Test organisms (inoculum):
soil

Study design

Total exposure duration:
28 d

Test conditions

Test temperature:
Room Temperature throughout Test Duration
Temperature [°C]
nominal measured
20 ± 2 19 - 23 (1)
(1) The temperature increased > 22 °C for about 162 hours.
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 – 0.9 %). Demineralised water was added as necessary.
Details on test conditions:
Control Untreated soil was tested under the same conditions as the test replicates.
Replicates Triplicates
Soil amount per replicate 400 g soil dry weight
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 5.3).
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 experimental starting.
Temperature 20 ± 2 °C (nominal)
Photoperiod Dark
Soil moisture content 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 – 0.9 %). Demineralised water was added as necessary.
Aeration The soil was incubated in the above mentioned boxes. The tops of the boxes were perforated to enable gas exchange.
Nominal and measured concentrations:
the nominal concentrations of 25.6, 64, 160, 400 and 1000 mg/kg soil dry weight. These concentrations correspond to the following
geometric mean measured concentrations: 3.76 – 6.30 – 12.9 – 25.4 – 53.1 mg/kg soil dry weight.
Reference substance (positive control):
yes
Remarks:
Cyanoguanidine

Results and discussion

Effect concentrationsopen allclose all
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
>= 53.1 mg/kg soil dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
47.5% active ingredient
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
>= 25.22 mg/kg soil dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
Based on 100% active ingredient
Basis for effect:
nitrate formation rate
Details on results:
The rates of nitrate formation were statistically significant higher in the geometric mean measured test item concentration 53.1 mg/kg DW compared to the untreated control after 7 and 28 days of exposure (Figure 1). Consequently, there was no evidence that exposure to Dissolvine GL-47-S inhibited the nitrogen transformation activity of soil microflora at the range of concentrations applied. The EC50 based on inhibition of nitrogen transformation processes could not be determined, as no inhibitory effects were observed at any test item concentration. The corresponding EC10 was ≥ 53.1 mg test item/kg soil dry weight, this concentration is equivalent to the nominal concentration of 1000 mg/kg soil dry weight.
Results with reference substance (positive control):
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 that used for studies with test items.

Table 18: 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 10.9 ± 0.27 23.5 ± 0.35 29.0 ± 1.35 38.1 ± 0.60
50 11.3 ± 0.67 13.1 ± 0.52 14.0 ± 0.06 26.5 ± 0.44
100 11.0 ± 0.27 12.9 ± 0.56 13.1 ± 1.04 11.6 ± 0.12

Table 19: Inhibition of Nitrate-N Formation Rates of the Reference Item Test
Nitrate-N Formation Rate
Test concentration [%] compared to Untreated Control
[mg/kg soil dry weight]
7 d 14 d 28 d
50 86* 85* 44*
100 85* 88* 98*
*) 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.
Reported statistics and error estimates:
Statistical analysis
One Way Analysis of Variances (ANOVA) and DUNNETT’S test were carried out for the determination of statistically significant differences of the nitrate formation rate at test end compared to control replicates. When running a One Way Analysis of Variances a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The α-value for ANOVA and DUNNETT’S test (acceptable probability of incorrectly concluding that there is a difference) is α = 0.05. For Nitrate formation rate on day 14 the Normality test failed, therefore a Kruskal-Wallis One Way analysis of variance on Ranks was carried out.

EC-values and confidence Since no inhibition of nitrate formation was encountered in any of intervals the Dissolvine GL-47-S (CAS No. 051981-21-6) treatments, it was not possible to determine inhibitory EC endpoints from the results of this study.
Software The data for the tables in the report were computer generated and have been rounded for presentation from the fully derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures.
Calculations were carried out using software
Excel, MICROSOFT CORPORATION
SigmaPlot, SPSS INC.
GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Any other information on results incl. tables

Measured Concentrations and Percentages of the nominal Concentrations of the Test Item

Sampling date

2015-07-01

2015-07-29

 

Start of analysis

2015-07-01*

2015-07-30

Nominal concentration

Dissolvine GL-47-S (CAS No.051981-21-6)

 

 

[mg/kg DW]

 

[mg/L]

(stock solution)

Meas. conc.

[mg/L]



%

Meas. conc.

[mg/kg]



%

Geometric mean measured test item concentration [mg/kg]2)

1000

70371

72562

103

1.60

1

53.1

 400

29019

31146

107

    4.65

1

   25.4

 160

11789

10761**

91

         3.011)

2

   12.9

   64.0

4719

4119**

87

         1.791)

3

       6.30

    25.6

1908

1683**

88

         1.601)

6

       3.76

Control

-

< SysQL

< SysQL

-

Measured concentration     = Measured concentration of the test item, dilution factor taken into account

*                           = Reinjected on 2015-07-02

**                          = Reanalysed on 2015-07-03, mean value of two replicates and two injections each

%                          = Percentage of nominal concentration of the test item

SysQL                     =System Quantification Limit(0.5 mg/L test item)

1)                          = Values < SysQL but > LOD were calculated and taken into account for Geometric mean calculation

2)                          = For Geometric mean calculation, nominal values at the start of the exposure (verified via stock solution analysis) were taken into account

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
All validity criteria specified by the test guidelines were fulfilled.
Conclusions:
The test is considered valid as all validity criteria have been fulfilled; Based on the test setup (GLP, Guideline study) and the extensive effort which has been paid to quantify the exposure not only at the start but also at the end of the study the results are considered to be reliable.
Executive summary:

The effects ofDissolvine GL-47-S(CAS No.051981-21-6)(Batch no.FC-C11836) on the metabolic activity of soil micro-organisms were determined according to OECD Guideline 216 (2000) atDr.U.Noack-Laboratorien, D-31157 Sarstedt, Germany from 2015-07-01 to 2015-07-30. 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 withDissolvine GL-47-S.

 

The test item was applied via demineralised water at the nominal concentrations of 25.6, 64, 160, 400 and 1000 mg/kg soil dry weight. These concentrations correspond to the following geometric mean measured concentrations: 3.76 – 6.30 – 12.9 – 25.4 – 53.1 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 withDissolvine GL-47-Sagainst 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 statistically significant higher in the geometric mean measured test item concentration 53.1 mg/kg DW compared to the untreated control after 7 and 28 days of exposure (Figure1). Consequently, there was no evidence that exposure toDissolvine GL-47-Sinhibited the nitrogen transformation activity of soil microflora at the range of concentrations applied. The EC50based on inhibition of nitrogen transformation processes could not be determined, as no inhibitory effects were observed at any test item concentration. The corresponding EC10was ≥ 53.1 mgDissolvine GL-47-S/kg soil dry weight, this concentration is equivalent to the nominal concentration of 1000 mg/kg soil dry weight.

 

The cause of the partly dose-related increase in nitrate formation observed is unknown. Partial breakdown of the test item may have released nitrogen for mineralisation. Further clarifications of the underlying mechanism are not possible without making substantial alterations to the study design that are beyond the requirements and purpose of the test guideline.

 

The determination of the concentrations ofthe test itemwas carried out via LC-MS.Evaluation was done from freshly prepared stock solutions on the day of application and from soil samples on day 28. The measured concentrations of the test itemin the stock solutions at the test initiation were in the range of 87 to 107 % of the nominal values. After 28 days, test item concentrations of 1 – 6 % of the nominal values were detected in the soil samples (Table 7). This finding suits quite well the already reported biodegradability of the test substance class (Ginkel, 2007). An extraction efficiency test revealed a sufficient general extraction potential of the method. Therefore, all analyses were done based on geometric mean measured test item concentrations.