Tall oil, maleated

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021 - 2022

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:

adopted January 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The following information concerning identity and composition of the test item was provided by the sponsor.
Name: Tall oil, maleated
CAS no.: 68152-93-2
EC no.: 268-859-6
Copy of Certificate of Analysis is attached to study report
Storage in Test Facility: The test item was stored in the test facility in a closed vessel at room temperature (20 ±5 °C), kept under inert gas.

Analytical monitoring:

yes

Remarks:

The analytical determination of nitrate was performed via IC following SANCO/3029/99 rev. 4 dated 11. July 2000.

Vehicle:

no

Details on preparation and application of test substrate:

For determination of the microbial biomass, 8 samples were taken from the soil. Four samples, each 10.0 g, were extracted using 0.5 M K2SO4 solution, shaken for 1 hour, centrifuged for 10 min (4500 rpm), filtered through nylon filter and the TOC (total organic carbon) of the filtrated solution was measured. The water content of the soil at this time point was measured.
Then, the 4 other samples, each 20.0 g, were fumigated using chloroform for 4 days and then extracted as described above. The water content of each sample was measured.
TOC Measurement: Analyses of the dissolved organic carbon were made by TC and IC measurement using the carbon analyser TOC multi N/C 2100S, Analytik Jena. Each sample was measured in duplicate or triplicate, respectively (depending on the variation between the measured values). The carbon analyser was calibrated every 6 months with freshly prepared reference solutions containing potassium hydrogen phthalate (TC), sodium hydrogen carbonate and sodium carbonate (IC). The quality control samples were measured on each measurement day. TOC (Total organic carbon) is determined as TC (total carbon) minus IC (inorganic carbon) stock solution.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

Temperature during study performance: 19.5 – 21.2 °C

Moisture:

Water holding capacity of soil was 35.2 % ±1.8%. Demineralised water was used to adjust the soil to a water content of 40 to 60 % of the maximum water holding capacity.

Organic carbon content (% dry weight):

0.65

Nitrogen content (% dry weight):

0.07

Nominal and measured concentrations:

10, 32, 100, 320 and 1000 mg/kg dry weight (nominal) were measured. The concentrations to be tested were based on a non GLP pre-test.

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

754.98 mg/kg soil dw

95% CI:

> 545.71 - <= 1 044.52

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

see below in tables

Results with reference substance (positive control):

no positive control measured

Reported statistics and error estimates:

see below

Analytical Determination

The nitrate content of the replicates was measured at the start and the end of the test via IC-determination. The measured concentrations for treatments and controls are given in the following tables:

Table Measured Nitrate Concentrations at 0 d

Nominal Concentration [mg/kg]	Replicate	Area [mAU*min]	Measured Concentration [mg/L]	QC recovery [%]	Calculated Concentration [mg/L]	Mean Calculated Concentration [mg/L]
QC sample (20 mg/L)	--	10.1548	20.1	100.7	--	--
	--	10.1731	20.2	100.9	--
Blank control	1	6.6180	13.3	--	13.3	13.31
		6.6016	13.3	--	13.3
	2	6.2484	12.6	--	12.6	12.38
		6.0101	12.1	--	12.1
	3	6.1730	12.5	--	12.5	12.46
		6.1678	12.5	--	12.5
Solvent control	4	6.0681	12.3	--	12.3	12.26
		6.0640	12.3	--	12.3
	5	5.9662	12.1	--	12.1	12.06
		5.9586	12.0	--	12.0
	6	5.9705	12.1	--	12.1	12.07
		5.9733	12.1	--	12.1
10	7	5.9958	12.1	--	12.1	12.14
		6.0115	12.2	--	12.2
	8	6.0422	12.2	--	12.2	12.21
		6.0438	12.2	--	12.2
	9	6.0782	12.3	--	12.3	12.26
		6.0568	12.2	--	12.2
32	10	5.9228	12.0	--	12.0	12.00
		5.9420	12.0	--	12.0
	11	6.0115	12.2	--	12.2	12.15
		6.0078	12.1	--	12.1
	12	5.9273	12.0	--	12.0	11.98
		5.9152	12.0	--	12.0
QC sample (20 mg/L)	--	10.1285	20.1	100.5	--	--
	--	10.1548	20.1	100.7	--	--
100	13	5.7628	11.7	--	11.7	11.66
		5.7556	11.7	--	11.7
	14	5.9821	12.1	--	12.1	11.91
		5.7910	11.7	--	11.7
	15	5.7327	11.6	--	11.6	11.62
		5.7355	11.6	--	11.6
320	16	5.9173	12.0	--	12.0	11.96
		5.9044	11.9	--	11.9
	17	5.8761	11.9	--	11.9	11.91
		5.9004	11.9	--	11.9
	18	5.8975	11.9	--	11.9	11.93
		5.8933	11.9	--	11.9
1000	19	5.7464	11.6	--	11.6	11.64
		5.7488	11.6	--	11.6
	20	5.7476	11.6	--	11.6	11.64
		5.7483	11.6	--	11.6
	21	5.7329	11.6	--	11.6	11.61
		5.7334	11.6	--	11.6
QC sample (20 mg/L)	--	10.1457	20.1	100.6	--	--
	--	10.1461	20.1	100.6	--

LOQ = 2.5 mg/L

 

Table Measured Nitrate Concentrations at 28 d

Nominal Concentration [mg/kg]	Replicate	Area [mAU*min]	Measured Concentration [mg/L]	QC recovery [%]	Calculated Concentration [mg/L]	Mean Calculated Concentration [mg/L]
QC sample (20 mg/L)	--	10.2091	20.2	101.2	--	--
	--	10.2192	20.3	101.3	--
Blank control	1	8.8222	17.6	--	17.6	17.57
		8.8216	17.6	--	17.6
	2	8.5323	17.0	--	17.0	17.02
		8.5432	17.0	--	17.0
	3	9.2141	18.3	--	18.3	18.35
		9.2419	18.4	--	18.4
Solvent control	4	9.0431	18.0	--	18.0	17.97
		9.0121	17.9	--	17.9
	5	9.0190	18.0	--	18.0	17.95
		9.0187	18.0	--	18.0
	6	8.4716	16.9	--	16.9	16.94
		8.5172	17.0	--	17.0
10	7	9.3841	18.7	--	18.7	18.65
		9.3802	18.6	--	18.6
	8	9.1583	18.2	--	18.2	18.23
		9.1719	18.2	--	18.2
	9	9.7484	19.4	--	19.4	19.37
		9.7570	19.4	--	19.4
32	10	10.7647	21.3	--	21.3	21.32
		10.7703	21.3	--	21.3
	11	10.7536	21.3	--	21.3	21.29
		10.7460	21.3	--	21.3
	12	10.6819	21.2	--	21.2	21.16
		10.6787	21.2	--	21.2
QC sample (20 mg/L)	--	10.2959	20.4	102.1	--	--
	--	10.2974	20.4	102.1	--	--
100	13	13.5011	26.6	--	26.6	26.59
		13.4929	26.6	--	26.6
	14	13.1666	26.0	--	26.0	25.90
		13.1137	25.8	--	25.8
	15	13.1602	25.9	--	25.9	25.96
		13.1865	26.0	--	26.0
320	16	8.8398	17.6	--	17.6	17.62
		8.8544	17.6	--	17.6
	17	8.3302	16.6	--	16.6	16.62
		8.3269	16.6	--	16.6
	18	8.6096	17.2	--	17.2	17.16
		8.6038	17.2	--	17.2
1000	19	3.6060	7.5	--	7.5	7.51
		3.6020	7.5	--	7.5
	20	3.3319	7.0	--	7.0	6.98
		3.3317	7.0	--	7.0
	21	3.4315	7.2	--	7.2	7.18
		3.4340	7.2	--	7.2
QC sample (20 mg/L)	--	10.2653	20.4	101.8	--	--
	--	10.2620	20.3	101.7	--

LOQ = 2.5 mg/L

 

Nitrate Concentration

The nitrate concentration at the start and the end of the test in mg/kg is stated in the following table.

Table Nitrate Concentration

Nominal Concentration in mg/kg dry matter	Replicate	Nitrate Concentration in mg/kg at day 0	Nitrate Concentration in mg/kg at day 28
Blank control	1	131.57	173.97
	2	122.37	168.53
	3	123.17	181.70
	Mean	125.70	174.73
	SD	5.09	6.62
	RSD	4.1	3.8
Solvent control	4	121.23	178.04
	5	119.26	177.84
	6	119.36	167.84
	Mean	119.95	174.57
	SD	1.11	5.84
	RSD	0.9	3.3
10	7	120.24	185.28
	8	120.93	181.11
	9	121.42	192.43
	Mean	120.86	186.27
	SD	0.60	5.73
	RSD	0.5	3.1
32	10	118.86	210.13
	11	120.35	209.84
	12	118.67	208.56
	Mean	119.29	209.51
	SD	0.92	0.84
	RSD	0.8	0.4
100	13	115.44	262.94
	14	117.91	256.12
	15	115.04	256.71
	Mean	116.13	258.59
	SD	1.56	3.78
	RSD	1.34	1.46
320	16	118.29	174.89
	17	117.80	164.96
	18	117.99	170.32
	Mean	118.03	170.06
	SD	0.25	4.97
	RSD	0.21	2.92
1000	19	115.11	74.52
	20	115.11	69.26
	21	114.81	71.24
	Mean	115.01	71.67
	SD	0.17	2.66
	RSD	0.15	3.71

SD = Standard Deviation

RSD = Relative Standard Deviation

 

Inhibition of Nitrate Production: In lack of significant difference between the blank and solvent control, both controls were pooled for the statistical determination. The mean and SD of inhibition of nitrate production per concentration in % compared to the pooled controls are stated in the table below.

Table Nitrate production in % compared to the pooled controls

Nominal Concentration in mg/kg dry matter	Mean inhibition of nitrate production in % compared to the pooled controls
Pooled controls	--
10	- 6.0
32	- 21.1
100	- 48.5
320	3.0
1000	59.1

Negative inhibition values indicate a stimulation of nitrate production compared to the pooled controls.

 

Statistics and Biological Results: Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.3.0.

 

The following results were determined: 28d-EC50: 750.10 mg/kg (confidence interval 538.67 mg/kg – 1044.50 mg/kg; This value was extrapolated by the program)

Validity criteria fulfilled:

yes

Conclusions:

The 28d-EC50 for nitrate formation inhibition was determined as 754.98 mg/kg dw (confidence interval 545.71 – 1044.52 mg/kg dw).

Executive summary:

One valid experiment was performed in this OECD 216 study to determine inhibition of nitrate formation through terrestrial microorganisms. The study was performed using 5 concentrations ranging from 10 to 1000 mg/kg nominal.

Sieved soil was exposed to the test item for 28 days. After 0 and 28 days, the nitrate content of the soil of each treatment was determined using IC-determination. A blank and solvent control was tested. Because of no significant difference between the blank and solvent control, both controls were pooled for the statistical determination.

The two highest test concentrations showed inhibition of nitrate production compared to the pooled controls.

The relative standard deviation between the replicates of the pooled controls was 3.2 % and indicated a small difference between the replicates. No observations were made which might cause doubts concerning the validity of the study outcome. Thus, the result of the test can be considered valid. No deviations from the Study Plan were recorded.

In deviation from the guidelines, the soil was stored in the laboratory under test conditions. As the storage takes place in the dark and without air contact, this deviation can be stated as uncritical.

Inhibition of nitrate formation was only seen at elevated concentrations and the 28d EC50 in this study was found as 754.98 mg/kg soil dw.

Description of key information

One valid experiment was performed according to OECD 216 investigating tall oil, maleated.
The study was performed using 5 concentrations ranging from 10 to 1000 mg/kg nominal. Sieved soil was exposed to the test item for 28 days. After 0 and 28 days, the nitrate content of the soil of each treatment was determined using IC-determination. A blank and solvent control were tested. Because of no significant difference between the blank and solvent control, both controls were pooled for the statistical determination.
The two highest test concentrations (320 and 1000 mg/kg soil dw) showed inhibition of nitrate production compared to the pooled controls. The following results were determined for the test item Tall oil, maleated: 28d-EC50 = 754.98 mg/kg dw (confidence interval 538.67 mg/kg – 1044.50 mg/kg).

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:

754.98 mg/kg soil dw