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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
Study period:
27 April to 25 May 2017
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)
Version / remarks:
OECD, 2000. OECD Guideline for the testing of chemicals, No. 216. Soil Microorganisms: Nitrogen Transformation Test. Adopted January 21, 2000.
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
No further details specified in the study report.
Analytical monitoring:
yes
Details on sampling:
In order to assess the influence of the test substance on the nitrification process, a 20-g dry weight subsample was taken from each replicate of each soil test group (control and test substance treated) at sampling intervals of 0, 7, 14, and 28 days.
Vehicle:
yes
Remarks:
acetone
Details on preparation and application of test substrate:
Stock Solution Preparation
A 50.3 mg/mL primary stock solution was prepared prior to exposure initiation by diluting 0.2514 g of decanoic acid, mixed esters with heptanoic acid, octanoic acid, and trimethylolpropane (Hatcol 1510) to a volume of 5.0 mL with acetone (CAS No. 67-64-1).
A 10.1 mg/mL secondary stock solution was prepared by diluting 1.0 mL of the 50.3 mg/mL primary stock to a final volume of 5.0 mL with acetone.
A 1.26 mg/mL secondary stock solution was prepared by diluting 0.150 mL of the 50.3 mg/mL primary stock to a final volume of 6.0 mL with acetone.

Dosing Procedure
For each test substance concentration and control, 10 g of quartz sand was weighed out and dosed with the appropriate dosing solution. The control sand was dosed with acetone to simulate stock application. In all cases, the solvent was allowed to evaporate off prior to mixing the dosed quartz sand into the soil.
Six 1-kg (dry weight) batches of soil were weighed out into 1-L beakers. One at a time, each 1-kg batch of soil was transferred to the bowl of a mechanical mixer and mixed with the appropriate batch of dosed quartz sand, alfalfa, and purified reagent water. From each mixed and treated batch of soil, three replicates of approximately 333 g (dry weight) soil each were weighed into individual 1-L glass bottles. The application proceeded according the to the dosing table listed below.
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
The samples were incubated at a temperature of 20 ± 2 °C in an environmental chamber. The temperature was monitored continuously with a minimum/maximum thermometer.
Moisture:
The moisture content of the soil samples was checked and, if necessary, adjusted on days 7, 14, 21, and 28.
The moisture content used for the definitive test was 9.36% (as dry weight basis).
Details on test conditions:
Test Soil
The Speyer 2.3 soil used for the test was collected by Lufa-Speyer in Germany on 10 February 2017. Over the past four years, no pesticides have been applied to the collection site; fertilizer was applied in 2014. The freshly-collected soil was taken from the top 20 cm, characterized by Lufa-Speyer, and allowed to dry slightly prior to being sieved through a 2-mm sieve. The soil was then shipped to Smithers Viscient in Wareham, Massachusetts, USA, where it was stored refrigerated.
The moisture content of the Speyer 2.3 soil upon arrival was 5.78% on a wet weight basis. The microbial biomass was determined at Smithers Viscient by fumigation/extraction methods (Brookes, et.al., 1990) to be 11.1 mg C/100 g soil (1.9% organic carbon).
The maximum water holding capacity (WHC) determined by Agvise Laboratories, Northwood, North Dakota was 23.4%. Approximately 11.5 mL of purified reagent water was added per 1000 g (dry weight) of soil to bring the WHC to 40% for the definitive test. The moisture content used for the definitive test was 9.36% (as dry weight basis).
The alfalfa (also known as Lucerne Meal) used to amend the test soil was obtained from Hanson Grain in Hanson, Massachusetts on 29 September 2011 and was determined to have a nitrogen content of 2.7% and a carbon content of 41.2% (equaling a ratio of approximately 1:15).

Dosing Procedure
For each test substance concentration and control, 10 g of quartz sand was weighed out and dosed with the appropriate dosing solution. The control sand was dosed with acetone to simulate stock application. In all cases, the solvent was allowed to evaporate off prior to mixing the dosed quartz sand into the soil.
Six 1-kg (dry weight) batches of soil were weighed out into 1-L beakers. One at a time, each 1-kg batch of soil was transferred to the bowl of a mechanical mixer and mixed with the appropriate batch of dosed quartz sand, alfalfa, and purified reagent water. From each mixed and treated batch of soil, three replicates of approximately 333 g (dry weight) soil each were weighed into individual 1-L glass bottles.

Test Conditions
The test flasks were covered with perforated parafilm, which allowed for air exchange and avoided loss in moisture. The samples were incubated at a temperature of 20 ± 2 °C in an environmental chamber. The temperature was monitored continuously with a minimum/maximum thermometer. The moisture content of the soil samples was checked and, if necessary, adjusted on days 7, 14, 21, and 28.

Test Procedures for Determination of NO3- Ions
In order to assess the influence of the test substance on the nitrification process, a 20-g dry weight subsample was taken from each replicate of each soil test group (control and test substance treated) at sampling intervals of 0, 7, 14, and 28 days. The subsamples were extracted with 100 mL of 0.1 M potassium chloride (KCl) for 60 minutes on a shaker table set at 150 rpm. After the extraction, the samples were centrifuged for 10 minutes at 1000 rpm. An aliquot of the supernatant was centrifuged at 10,000 rpm for 5 minutes and transferred to an HPLC vial.
Nominal and measured concentrations:
The test was performed at concentrations of 1.2, 3.7, 11, 33, and 100 mg/kg dry weight soil and an untreated control.
Reference substance (positive control):
no
Key result
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
Details on results:
Test Conditions
During the test period, the temperature in the incubation chamber containing the soil samples was 20 to 21°C. The soil moisture content was maintained at approximately 40% of the maximum water holding capacity (WHC) throughout the test.

Nitrogen Transformation
The results showed that the soil microorganisms of the control, and the 1.2, 3.7, 11, 33, and 100 mg/kg treated samples transformed alfalfa-bound nitrogen into nitrate-bound nitrogen.

Validity of the Study
Variations in the quantity of nitrate (mg NO3-/kg dry soil) between the replicate nitrate determination control samples in this test ranged from 0.81 to 2.94% and meet the requirement of less than 15% variation. Therefore, the validity criterion was fulfilled.
Results with reference substance (positive control):
Reference substance not utilised for this study.
Reported statistics and error estimates:
The day 14 nitrate values in the 1.2 mg/kg treatment level (41.08, 41.81, and 54.72 mg NO3-/L) were evaluated by a Q-Test (Dean and Dixon, 1951) and determined that 54.72 mg NO3-/L (replicate C) was an outlier. Therefore, it was excluded from further calculations.
Differences in the nitrate formation rate between the control and treated soils were tested for significance using the ANOVA and Dunnett’s Multiple Comparison Test (U.S. EPA, 2000), statistical programs in CETIS (Ives, 2013). No statistically significant effects could be determined from the differences in the nitrate formation rate of control and treated soils.
The quantity of nitrate (mg NO3-/kg dry weight soil) formed in the treated and control soil samples after 28 days of incubation were analyzed using a linear interpolation model. The EC10, EC25, and EC50 values are determined to be 0.795, 39.7, and >100 mg/kg respectively.

 Nitrate concentrations measured during the soil microorganisms study with decanoic acid, mixed esters and heptanoic acid, octanoic acid, and trimethylolpropane (Hatcol 1510).

Treatment Group

Day 0

(mg NO3-/kg dry soil)

Day 7

(mg NO3-/kg dry soil)

Day 14

(mg NO3-/kg dry soil)

Day 28

(mg NO3-/kg dry soil)

Control

 

 

 

Mean ± SDa

%CVb

177.05

174.95

171.15

 

174.38 ± 2.99

1.71

210.70

205.15

210.35

 

208.73 ± 3.11

1.49

243.50

247.45

245.75

 

245.57 ± 1.98

0.81

275.75

292.10

287.00

 

284.95 ± 8.37

2.94

Decanoic acid, mixed esters with heptanoic acid, octanoic acid, and trimethylolpropane (Hatcol 1510)

1.2 mg/kg

 

 

 

Mean ± SD

150.50

148.50

150.00

 

149.67 ± 1.04

168.80

164.50

169.90

 

167.73 ± 2.85

205.40

209.05

-c

 

207.23 ± 2.58

246.55

225.25

235.40

 

235.73 ± 10.65

3.7 mg/kg

 

 

 

Mean ± SD

160.85

163.50

164.60

 

162.98 ± 1.93

191.60

186.55

179.75

 

185.97 ± 5.95

207.10

225.55

229.20

 

220.62 ± 11.85

256.20

256.30

259.50

 

257.33 ± 1.88

11 mg/kg

 

 

 

Mean ± SD

140.40

152.25

143.55

 

142.07 ± 1.58

149.50

146.95

141.35

 

145.93 ± 4.17

195.40

196.10

190.65

 

194.05 ± 2.97

246.94

23.10

236.75

 

237.93 ± 8.49

33 mg/kg

 

 

 

Mean ± SD

141.10

140.30

140.75

 

140.72 ± 0.40

144.80

143.70

145.35

 

144.62 ± 0.84

186.05

186.10

181.35

 

184.50 ± 2.73

234.80

222.75

215.80

 

224.45 ± 9.61

100 mg/kg

 

 

 

Mean ± SD

109.15

111.30

112.30

 

110.92 ± 1.61

86.35

85.35

90.80

 

87.50 ± 2.90

128.15

128.85

131.40

 

129.47 ± 1.71

154.80

156.65

166.85

 

159.43 ± 6.49

aSD = Standard Deviation

bCV = Coefficient of Variation

cThis replicate was considered an outlier and was not included in the mean value

NOTE: All mean values presented in the table were calculated with unrounded numbers.

 

Nitrate transformation rates measured during the soil microorganisms study with decanoic acid, mixed esters with heptanoic acid, octanoic acid, and trimethylolpropane (Hatcol 1510).

Treatment Group

Day 7

(mg NO3-/kg dry soil/day)

Day 14

(mg NO3-/kg dry soil/day)

Day 28

(mg NO3-/kg dry soil/day)

Control

 

 

 

Mean ± SDa

4.81

4.31

5.60

 

4.91 ± 0.65

4.69

6.04

5.06

 

5.26 ± 0.70

2.30

3.19

2.95

 

2.81 ± 0.46

Decanoic acid, mixed esters with heptanoic acid, octanoic acid, and trimethylolpropane (Hatcol 1510)

1.2 mg/kg

 

 

 

Mean ± SD

% difference from control

2.61

2.29

2.84

 

2.58 ± 0.28

-47.40

5.23

6.36

-b

 

5.80 ± 0.80

10.16

2.94

1.16

3.12

 

2.41 ± 1.08

-14.50

3.7 mg/kg

 

 

 

Mean ± SD

% difference from control

4.39

3.29

2.16

 

3.28 ± 1.11

-33.09

2.21

5.57

7.06

 

4.95 ± 2.48

-5.93

3.51

2.20

2.16

 

2.62 ± 0.77

-6.77

11 mg/kg

 

 

 

Mean ± SD

% difference from control

1.30

0.67

-0.31

 

0.55 ± 0.81

-88.74

6.56

7.02

7.04

 

6.87 ± 0.27

30.63

3.68

2.43

3.29

 

3.13 ± 0.64

11.43

33 mg/kg

 

 

 

Mean ± SD

% difference from control

0.52

0.49

0.66

 

0.56 ± 0.09

-88.65

5.89

6.06

5.14

 

5.70 ± 0.49

8.28

3.48

2.62

2.46

 

2.85 ± 0.55

1.44

100 mg/kg

 

 

 

Mean ± SD

% difference from control

-3.26

-3.71

-3.07

 

-3.35 ± 0.33

-168.17

5.97

6.21

5.80

 

6.00 ± 0.21

13.94

1.90

1.99

2.53

 

2.14 ± 0.34

-23.91

aSD = Standard Deviation

bThis replicate was considered an outlier and was not included in the mean value.

NOTE: All mean values presented in the table were calculated with unrounded numbers.

Validity criteria fulfilled:
yes
Conclusions:
Based on the differences in nitrate transformation rates for 28 days of exposure, it can be concluded that decanoic acid, mixed esters with heptanoic acid, octanoic acid, and trimethylolpropane (Hatcol 1510) has no long-term effects on the nitrification rates of soil microbial life at concentrations ≤ 100 mg/kg dry soil.
Executive summary:

PROTOCOL TITLE: Decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) - Determination of the Effects on Soil Microorganism Activity Following OECD Guideline 216

SMITHERS VISCIENT STUDY NUMBER: 41.6249

TEST SUBSTANCE: Decanoic acid, mixed esters with heptanoic acid, octanoic acid, and trimethylolpropane (Hatcol 1510), Lot No. 2015189219 (TR269-131), CAS No./EC No. 68130-53-0/268-596-7, reported to have a purity of 100%, was received from Chemtura Corporation on 13 March 2017.

DEFINITIVE TEST DATES: The test incubation period was conducted between 27 April and 25 May 2017

TEST SOIL: Speyer 2.3 (sandy loam), Speyer, Germany; Organic carbon content: 0.76%; Maximum water holding capacity: 23.4%; Microbial biomass: 1.9% of total organic carbon

TEST CONDITIONS: 20.0 ± 2 °C

TEST CONCENTRATIONS: Control, 1.2, 3.7, 11, 33, and 100 mg/kg

PARAMETER MEASURED: Nitrogen transformation (Nitrification)

SAMPLING INTERVALS: Days 0, 7, 14, and 28

 

RESULTS: The mean NO3- concentrations in the control and treated soils are presented in the following table.

Concentration (mg/kg)

Day 0

(mg NO3-/kg dry soil)

Day 7

(mg NO3-/kg dry soil)

Day 14

(mg NO3-/kg dry soil)

Day 28

(mg NO3-/kg dry soil)

Control

174.38

208.73

245.57

284.95

1.2

149.67

167.73

207.23

235.73

3.7

162.98

185.97

220.62

257.33

11

142.07

145.93

194.05

237.93

33

140.72

144.62

184.50

224.45

100

110.92

87.85

129.47

159.43

 

These results showed that the soil microorganisms of the control and the 1.2, 3.7, 11, 33 and 100 mg/kg treated samples transformed alfalfa-bound nitrogen into nitrate-bound nitrogen.

 

The mean nitrate transformation rates and the percent difference from control values are presented in the following table.

Concentration (mg/kg)

Day 7 Rate

(mg NO3-/kg/day)

% Difference from Control

Day 14 Rate

(mg NO3-/kg/day)

% Difference from Control

Day 28 Rate

(mg NO3-/kg/day)

% Difference from Control

Control

4.91

NA

5.26

NA

2.81

NA

1.2

2.58

-47.40

5.80

10.16

2.41

-14.50

3.7

3.28

-33.09

4.95

-5.93

2.62

-6.77

11

0.55

-88.74

6.87

30.63

3.13

11.43

33

0.56

-88.65

5.70

8.28

2.85

1.44

100

-3.35

-168.17

6.00

13.94

2.14

-23.91

NA = Not Applicable

 

CONCLUSION: Based on the differences in nitrate transformation rates for 28 days of exposure, it can be concluded that decanoic acid, mixed esters with heptanoic acid, octanoic acid, and trimethylolpropane (Hatcol 1510) has no long-term effects on the nitrification rates of soil microbial life at concentrations ≤ 100 mg/kg dry soil. 

Description of key information

Decanoic acid, mixed esters with heptanoic acid, octanoic acid, and trimethylolpropane (Hatcol 1510) has no long-term effects on the nitrification rates of soil microbial life at concentrations ≤ 100 mg/kg dry soil. 

Key value for chemical safety assessment

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

Additional information

PROTOCOL TITLE: Decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) - Determination of the Effects on Soil Microorganism Activity Following OECD Guideline 216

TEST SOIL: Speyer 2.3 (sandy loam), Speyer, Germany; Organic carbon content: 0.76%; Maximum water holding capacity: 23.4%; Microbial biomass: 1.9% of total organic carbon

TEST CONDITIONS: 20.0 ± 2 °C

TEST CONCENTRATIONS: Control, 1.2, 3.7, 11, 33, and 100 mg/kg

PARAMETER MEASURED: Nitrogen transformation (Nitrification)

SAMPLING INTERVALS: Days 0, 7, 14, and 28

 

RESULTS: The mean NO3- concentrations in the control and treated soils are presented in the following table.

Concentration (mg/kg)

Day 0

(mg NO3-/kg dry soil)

Day 7

(mg NO3-/kg dry soil)

Day 14

(mg NO3-/kg dry soil)

Day 28

(mg NO3-/kg dry soil)

Control

174.38

208.73

245.57

284.95

1.2

149.67

167.73

207.23

235.73

3.7

162.98

185.97

220.62

257.33

11

142.07

145.93

194.05

237.93

33

140.72

144.62

184.50

224.45

100

110.92

87.85

129.47

159.43

 

These results showed that the soil microorganisms of the control and the 1.2, 3.7, 11, 33 and 100 mg/kg treated samples transformed alfalfa-bound nitrogen into nitrate-bound nitrogen.

 

The mean nitrate transformation rates and the percent difference from control values are presented in the following table.

Concentration (mg/kg)

Day 7 Rate (mg NO3-/kg/day)

% Difference from Control

Day 14 Rate (mg NO3-/kg/day)

% Difference from Control

Day 28 Rate (mg NO3-/kg/day)

% Difference from Control

Control

4.91

NA

5.26

NA

2.81

NA

1.2

2.58

-47.40

5.80

10.16

2.41

-14.50

3.7

3.28

-33.09

4.95

-5.93

2.62

-6.77

11

0.55

-88.74

6.87

30.63

3.13

11.43

33

0.56

-88.65

5.70

8.28

2.85

1.44

100

-3.35

-168.17

6.00

13.94

2.14

-23.91

NA = Not Applicable

 

CONCLUSION: Based on the differences in nitrate transformation rates for 28 days of exposure, it can be concluded that decanoic acid, mixed esters with heptanoic acid, octanoic acid, and trimethylolpropane (Hatcol 1510) has no long-term effects on the nitrification rates of soil microbial life at concentrations ≤ 100 mg/kg dry soil.