2-ethylhexanoic acid, manganese salt

Toxicity to soil microorganisms

Administrative data

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed to an ISO guideline and to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

ISO 14238

Deviations:

yes

Remarks:

An extra endpoint was included in this study compared to the ISO test as substrate (ammonia) may be exhausted after 28 days.

Principles of method if other than guideline:

The test performed in this study is the ISO 14238 soil quality test in which ammonium is added to the soil before incubation. The percentage nitrate, relative to the added ammonium, is measured after 28 days incubation. The endpoint is called Substrate Induced Nitrification (SIN). An extra endpoint was included in this study compared to the ISO test as substrate (ammonia) may be exhausted after 28 days. The potential Nitrification Rate (PNR), which is the nitrification rate at unlimited substrate (NH4+) availability, was measured within 9 days incubation. This adjustment in incubation time obtains a larger sensitivity to added metals (Smolders et al., 2001).

GLP compliance:

yes

Remarks:

K.U.Leuven

Test material

Test material information

Constituent 1

Reference substance name:

manganese chloride

IUPAC Name:

manganese chloride

Details on test material:

- Name of test material (as cited in study report): Manganese chloride
- Analytical purity: Not reported
- Impurities (identity and concentrations): Not reported
- Composition of test material, percentage of components:Not reported
- Storage condition of test material:Not reported
- Other: MnCl2 was supplied by the Manganese Reach Administration Association.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Total metal concentrations in soil were determined after the post-spiking incubation time for both spiked and control soils.

Test substrate

Vehicle:

yes

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Other: MnCl2 spiked soils were equilibrated for 7 days. Afterwards, soils were amended with 100 mg NH4-N/kg fresh soil using a stock solution containing 80 mg (NH4)2SO4/mL (Smolders et al., 2001).

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: All soils were air dried at 25°C and sieved to <4mm. Soil samples were preincubated at a moisture content equivalent to 50% of pF2 for 7 days at 20°.Uncontaminated soils were spiked with MnCl2 to seven concentrations (control plus six treatments; 0, 0.01, 0.032, 0.1, 0.32, 1 and 3.2 x CEC;). Added Mn concentrations in corresponding treatments differ between the 3 soils as their CEC values increase from 6 to 21 cmolc/kg. Additional deionised water was added together with the spike solution to adjust the soil moisture content to 75% of pF2.0. All soils were thoroughly mixed after amendments using laboratory spoons. These spiked and preincubated soils was used for all microbial assays.

Test organisms

Test organisms (inoculum):

soil

Study design

Total exposure duration:

28 d

Test conditions

Test temperature:

20°c

Moisture:

Deionised water was added together with the spike solution to adjust the soil moisture content to 75% of pF 2.0.

Details on test conditions:

TEST SYSTEM
- No. of replicates per concentration: 3
- No. of replicates per control: 3

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site : Soil 1: Kasterlee (Belgium), Soil 2: Ter Munck (Belgium), Soil 3: Nagyhorcsok (Hungary)
- Depth of sampling: Not reported
- Soil texture
- % sand: Soil 1- 79%, Soil 2- 8%, Soil 3- 13%
- % silt: Soil 1- 9%, Soil 2- 70%, Soil 3- 54%
- % clay: Soil 1- 2%, Soil 2- 12%, Soil 3- 18%
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight):
- Maximum water holding capacity (in % dry weight):Soil 1 - pF0 -47%, pF1.8 -31, pF2- 27. Soil 2- pF0 -43%, pF1.8 -34, pF2-34. Soil 3-pF0 -48%, pF1.8 -35, pF2- 34
- Cation exchange capacity (mmol/kg): Soil 1-6.3, Soil 2-12.2 , Soil 3-24.8
- Storage : Air dry, sieved soils were stored in 60 litre plastic drums.

DETAILS OF PREINCUBATION OF SOIL (if any):
MnCl2 spiked soils were equilibrated for 7 days. Afterwards, soils were ammended with 100 mg NH4 -N/kg freash soil were thoroughly mixed, and incubated at 20°c in dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The percentage nitrate, relative to the added ammonium, was measured after 28 days incubation. The endpoint is called the Substrate Induced Nitrification (SIN). The potential Nitrification Rate(PNR), which is the nitrification rate at unlimited substrate (NH4+) availability, was measured within 9 days incubation.

VEHICLE CONTROL PERFORMED: no


RANGE-FINDING STUDY
- Range finding study
- Test concentrations: Uncontaminated soils were spiked with MnCl2 to seven concentrations (control plus six treatments; 0, 0.01, 0.032, 0.1, 0.32, 1 and 3.2 x CEC;). Added Mn concentrations in corresponding treatments differ between the 3 soils as their CEC values increase from 6 to 21 cmolc/kg
- Results used to determine the conditions for the definitive study: The rationale behind this spiking procedure (Mn addition as a fraction of the CEC) was based on previous research on metal toxicity in the lab showing strong positive correlations between metal toxicity (ECx values) and soil CEC i.e. soils with high CEC generally have higher ECx values than soil with low CEC. Therefore it was postulated that higher Mn additions were required in soil with high CEC compared to soil with low CEC to have a similar ECx values.

Nominal and measured concentrations:

Nominal Concentration
Soil 1- 0, 17, 55, 170, 545, 1703, 5450 mg/kg
Soil 2- 0, 34, 107, 335, 1072, 3351, 10724 mg/kg
Soil 3- 0, 68, 218, 681, 2180, 6813, 21800 mg/kg

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Duration:

28 h

Dose descriptor:

NOEC

Effect conc.:

207 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: Mn2+

Basis for effect:

nitrate formation rate

Details on results:

Not reported

Results with reference substance (positive control):

Not reported

Reported statistics and error estimates:

Statistical Data Analysis

Dose-response curves were fitted by a log-logistic model (Doelman and Haanstra, 1989) using the Maquardt Method (proc NLIN, SAS® 9.1; Cary, NC, USA):

Y = c/[1 + exp(b(x-a))]

With y = response variable, c= response in the control treatment, b= slope parameter, x= logarithm of the added Mn concentration and a = logarithm of the EC50 value. The model was adjusted to account for hormesis effects (Schabenberger et al., 1999; Van Ewijk and Hoekstra, 1993). The ‘dose’ in this model is the added Mn concentration (nominal concentration); with the dose in the control soil attributed a very small value (e.g. 0.001 mg Mn/kg). The EC50 values and their 95% confidence interval, expressed as added Mo, are predicted from the appropriate parameters of the curve. The EC50 values, expressed as ‘total Mn’, are calculated from the sum of background Mn concentration of a soil and corresponding EC50 (expressed as added Mn). No observed Effect Concentration (NOEC) are the highest Mn concentration in the soil at which no significant adverse effects on plant/ microbial assay were observed compared to the control soil (P> 0.05), and Lowest Observed Effect Concentrations (LOEC) are the lowest Mn concentration in the soil at which a significant adverse effect on plant/ microbial assay were observed compared to the control soil (P<0.05). Significant effects were determined by ANOVA (Dennett’s test). The threshold values (NOEC, LOEC and EC50) were determined in terms of soil Mn (mg/kg).

Any other information on results incl. tables

Potential Nitrification Rate (PNR) in freshly Mn spiked soils:

The PNR, measured from the increase in nitrate over time, was between 3.8 and 12.2 mg NO3- N/kg soil/d for the control soils, and decreased significantly with increasing MnCl2. The nitrification rate in the water soils was positively correlated with soil pH. Indeed, it has frequently been shown that the soil nitrification process is sensitive to soil pH ( Dancer et al., 1973; Smolders et al., 2001), suggesting that other factors besides the Mn concentration are also influential (e.g. soil pH, soil ionic strength (Sindhu and Cornfield 1967). Nevertheless, pH only slightly decreases upon MnCl2 addition to soil (Table 7 ). As a result, the inhibition of the soil nitrification process with increasing MnCl2 additions cannot be attributed to pH effects.

Substrate Induced Nitrification (SIN) in freshly Mn spiked soils:

After 28 days incubation the % age added ammonia that was nitrified (SIN), was measured. Almost all ammonia was nitrified in the control and low Mn contaminated treatments of the three soils. A significant hormesis effect was observed in the soil 3. Toxic effects of Mn were significant at added Mn concentrations of 0.32xCEC for the three soils. The confidence intervals for the EC10 and EC50 values are large. This is due to the steep decrease of the SIN with increasing Mn doses. At added Mn concentrations of 0.1xCEC, the SIN was 100% for the 3 soils, while at added Mn concentrations of 0.32xCEC the response was reduced by 50% in soil 1 and by almost 100% in soil 2. For the soil 3, no confidence intervals was derived due to the continuous increase of the SIN to the 0.32x CEC treatment at which no SIN was observed anymore.

Table 8. Soil Mn toxicity threshold for PNR in freshly spiked soils. Thresholds are expressed as added + background Mn and the Confidence Intervals (CI) of EC10 and EC50 are given.

 

Soil	Background Mn Conc. (mg/kg)	PNR (mg N/kg/d)	NOEC (mg/kg)	EC10 (mg/kg)	95% CI	EC50 (mg/kg)	95% CI
1	152	5.4	207	232	185-348	360	294-456
2	461	8.0	796	757	462->11185	812	502-3456
3	722	9.2	790	739	724-855	927	798-1278

 

Table 9. Soil Mn toxicity thresholds for Substrate Induced Nitrification (SIN, %) in freshly spiked soils. Thresholds are expressed as added + background Mn and the Confidence Intervals (CI) of EC10 and EC50 are given

 

Soil		Background Mn Conc. (mg/kg)		SIN (% substr. Used)		NOEC (mg/kg)		EC10 (mg/kg)		95% CI		EC50 (mg/kg)		95% CI
1		152		92		322		281		190-588		647		441-1000
2		461		93		796		1108		461->11185		1188		461->11185
3		722		74		1403		2571		nd		2593		nd

 

Table 10. Potential Nitrification Rate Calculated between 0-7 days after (NH₄)₂SO₄application in Mn freshly spiked soils.

  

Potential Nitrification Rate (PNR) in mg NO3- N/kg/day
Added Mn (mg/kg)	Soil 1	Added Mn (mg/kg)	Soil 2	Added Mn (mg/kg)	Soil 3
0	3.79	0	8.54	0	9.88
0	5.46	0	9.62	0	5.54
0	6.94	0	5.91	0	12.17
17	4.97	34	8.42	68	7.38
17	3.35	34	1.20	68	5.48
17	4.19	34	8.27	68	11.12
55	4.27	107	13.88	218	1.99
55	5.43	107	10.69	218	2.74
55	4.88	107	7.31	218	2.45
170	1.67	335	6.78	681	4.13
170	3.63	335	3.83	681	5.57
170	3.55	335	5.28	681	3.38
545	0.78	1072	<0.10	2180	0.57
545	<0.10	1072	<0.10	2180	0.99
545	0.62	1072	<0.10	2180	0.40
1703	<0.10	3351	<0.10	6813	<0.10
1703	<0.10	3351	<0.10	6813	<0.10
1703	<0.10	3351	<0.10	6813	<0.10
5450	<0.10	10724	<0.10	21800	<0.10
5450	<0.10	10724	<0.10	21800	<0.10
5450	<0.10	10724	<0.10	21800	<0.10

  

Table 11. Substrate Induced Nitrification calculated between 0-28 days after (NH₄)₂SO₄application in Mn spiked soils.

 

Potential Nitrification Rate (PNR) in mg NO3- N/kg/day
Added Mn (mg/kg)	Soil 1	Added Mn (mg/kg)	Soil 2	Added Mn (mg/kg)	Soil 3
0	70.08	0	67.68	0	63.41
0	106.85	0	124.70	0	82.37
0	100.13	0	87.12	0	77.09
17	86.16	34	97.30	68	97.39
17	118.13	34	100.61	68	65.90
17	114.29	34	116.83	68	58.51
55	95.38	107	84.67	218	110.21
55	109.58	107	118.13	218	56.69
55	119.95	107	97.10	218	58.56
170	66.72	335	106.18	681	161.28
170	58.51	335	105.12	681	141.94
170	86.78	335	134.78	681	130.46
545	46.51	1072	<0.10	2180	2.50
545	75.84	1072	<0.10	2180	2.35
545	50.02	1072	<0.10	2180	0.48
1703	<0.10	3351	<0.10	6813	<0.10
1703	<0.10	3351	<0.10	6813	<0.10
1703	<0.10	3351	<0.10	6813	<0.10
5450	<0.10	10724	<0.10	21800	<0.10
5450	<0.10	10724	<0.10	21800	<0.10
5450	<0.10	10724	<0.10	21800	<0.10

 

Applicant's summary and conclusion

Conclusions:

Nitrification rate is significantly affected by MnCl2 spiking. The toxicity thresholds varied between soils and generally increased with an increasing background Mn concentration. The lowest NOEC was 207 mg Mn/kg. The read-across from MnCl2 to MnSO4 is justified on the basis that both are very soluble in water, both have the same Mn2+ cation, and in the case of ecotoxicity neither the chloride or sulphate anions are considered to have any influence on the effective toxicity of Mn2+ or any toxicity in their own right, so the anions can be disregarded. Therefore any effect will be related to the Mn2+ cation, and the data from MnCl2 ecotoxicity tests is regarded as a suitable surrogate for read-across.