Molybdic acid

Bioaccumulation: terrestrial

Administrative data

Endpoint:

bioaccumulation: terrestrial

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Molybdenum levels in earthworms were measured in the surviving animals exposed to different soils with varying Mo concentrations. Data on Mo-levels in the soils were measured. No standard protocol was used.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Reference 2

Reference Type:

publication

Title:

The bioaccumulation of Molybdenum in the earthworm Eisenia andrei: Influence of soil properties and ageing

Author:

van Gestel CAM, Diez-Ortiz D, Borgman E, Verweij RA

Year:

2011

Bibliographic source:

Chemostphere 82, 1614-1619

Materials and methods

Principles of method if other than guideline:

Mo concentration was measured in surviving earthworms from a standard OECD 222 toxicity test with Eisenia andrei. BAFs for the accumulation of Mo in earthworms were calculated by dividing concentrations in the surviving animals by measured total concentrations in the test soils.

GLP compliance:

no

Remarks:

The study was carried out in compliance with the principles of good laboratory practice.

Test material

Test material information

Constituent 1

Reference substance name:

Sodium molybdate dihydrate

IUPAC Name:

Sodium molybdate dihydrate

Constituent 2

Reference substance name:

10102-40-6

Cas Number:

10102-40-6

IUPAC Name:

10102-40-6

Constituent 3

Reference substance name:

not available for the tested dihydrate

IUPAC Name:

not available for the tested dihydrate

Details on test material:

Name of test material (as cited in study report): Sodium Molybdate Dihydrate
- Molecular formula (if other than submission substance): Na2MoO4⋅2H2O
- Molecular weight (if other than submission substance): 241.95 g/mol
- Physical state: solid (white crystalline powder)
- Analytical purity: 99.9% (dihydrate form) based on an analysed Molybdenum content of 39.6%
- Expiration date of the lot/batch: 31 December 2008
- Storage condition of test material: at room temperature (10-30 degrees Celsius) in the dark, dry
- no further significant details stated

Information was not reported in the report, but tests were conducted with the same lot of test substance that was used in other test programs that were commissioned by IMOA

Sampling and analysis

Details on sampling:

For the determination of total and extractable metal concentrations and soil pH, for each soil and test concentration, two replicate test jars (containing approximately 30 g moist soil) were included. These jars did not receive animals and were sampled at the end of the toxicity tests, so approximately 4.5 weeks after spiking the soils.

Test substrate

Details on preparation and application of test substrate:

Solutions of sodium molybdate dihydrate in water were prepared and mixed with the soil. After adding Mo to the soils, moisture content was adjusted to 50% of the WHCmax. Soils were mixed with a household mixer for at least 2 minutes to obtain an as homogeneous as possible distribution of the Mo through the soil. Controls received water only. The freshly spiked soils were equilibrated for 4-6 days before starting exposure of the test organisms.

In addition, 3 sieved soils (soils 4, 5 and 6) were spiked with sodium molybdate (20 kg per soil per treatment was mixed with sodium molybdate in a concrete mill) and aged for 6 or 11 months on the field with free drainage prior to toxicity testing. The total Mo content was measured before the pots were placed on the field and after 6 and 11 months ageing. When the ageing period was finished, the soils were dried and sieved again. Before starting the toxicity tests, moisture content was adjusted to 50% of WHCmax.

Test organisms

Test organisms (species):

Eisenia sp.

Details on test organisms:

Earthworms of the species Eisenia andrei were obtained. The earthworms were cultured in a substrate of potting soil and peat, and fed abundantly with horse manure. the horse manure was obtained from healthy horses that were not recently treated with any pharmaceuticals. in the experiments, adult worms with fully developed clitellum were used.

Study design

Total exposure / uptake duration:

28 d

Total depuration duration:

1 d

Test conditions

Test temperature:

20°C

pH:

pH was soil dependent
soil 1 : 4.4
soil 2 : 5.0
soil 3 : 5.2
soil 4 : 5.2
soil 5 : 6.3
soil 6 : 6.7
soil 7 : 6.8
soil 8 : 7.3
soil 9 : 7.6
soil 10 : 7.8
soil 11: 7.0

TOC:

Organic carbon %
Soil 1: 30.7
Soil 2: 2.0
Soil 3: 2.8
Soil 4: 1.8
Soil 5: 3.6
Soil 6: 0.9
Soil 7: 0.6
Soil 8: 2.8
Soil 9: 2.7
Soil 10: 3.6
Soil 11: 5.8

Moisture:

Water content (% Mass) at 50% maximum Water Holding Capacity
soil 1 : 78.5
soil 2 : 18.5
soil 3 : 23.5
soil 4 : 23.0
soil 5 : 34.5
soil 6 : 21.5
soil 7 : 20.5
soil 8 : 21.0
soil 9 : 24.0
soil 10 : 22.0

Details on test conditions:

Tests with the earthworms included a 4-week exposure period of adult animals.
Four replicate test containers were used for each Mo concentration and control, containing approx. 500 g soil (dry weight equivalent), with 10 earthworms randomly being assigned to each container. At the start of the test, each batch of 10 animals was weighed, and 40 animals were weighed individually to determine the range of starting weights. Mean individual masses (+/- standard deviation; n=40) of the earthworms used in the different tests ranged between 303+/-73 mg and 415+/-79 mg. Test containers were incubated in climate room at 20 degrees Celsius, and constant illumination.
After introduction of the earthworms a small amount of finely ground and moistened horse dung (5g) was introduced as a food source. The food was placed in a small hole in the middle of the test soil as described by van Gestel et al., 1989. During the tests, atleast once a week all test containers were opened to aerate the test soils. In addition, once a week all test containers were weighed to correct for water loss and additional food was added if required.
After 4 weeks of incubation, test containers were emptied in a tray and surviving adults were collected by hand sorting and weighed (per test container). To determine bioaccumulation, surviving earthworms were analysed for internal Mo concentrations. For this analysis, test animals were placed on moist filter paper for one night to void their gut contents. After weighing they were freeze dried and digested using the same acid mixture and Teflon bombs as described for the soil samples. for each test concentration and soil type, if possible, 3-4 animals were analysed.

Soil samples were obtained from the plough layer (10-20 cm) of 8 agricultural soils and from the surface horizons after clearance of litter of 2 non-agricultural soils. After sampling, soils were air-dried, sieved through 4 mm and stored in drums at ambient temperature until use.

Nr † Location Country Soil type ‡ Land use Coordinates
1 Zegveld The Netherlands Histosol Grassland 52°07’11’’ N, 4°49’02’’ E
2 Kövlinge Sweden Dystric Regosol Arable land 55°47’38’’ N, 13°06’37’’ E
3 Kasterlee Belgium Haplic podzol Arable land 51°14’34’’ N, 4°58’37’’ E
4 Zwijnaarde Belgium Haplic podzol Arable land 50°59’54’’ N, 3°42’11’’ E
5 Woburn United Kingdom Dystric Cambisol Grassland 52°00’51’’ N, 0°35’48’’ W
6 Ter Munck Belgium Haplic Luvisol Arable land 50°52’42’’ N, 4°39’24’’ E
7 Souli Greece Chromic Luvisol Arable land 37°59’27’’ N, 22°39’32’’ E
8 Rots France Haplic Luvisol Arable land 49°12’15’’ N, 0°29’25’’ W
9 Nagyhörcsök Hungary Chernozem Arable land 46°54’25’’ N, 18°31’22’’ E
10 Guadelajara Spain Calcic Cambisol Olive orchard 40°37’02’’ N, 3°09’07’’ W
† Numbering according to pH
‡ Soil classification according to World Reference Base (FAO, ISRIC and ISSS, 1998).


Soil pH Organic C sand silt clay CEC at pHsoil Feox Mo Water content
0.01 M CaCl2 pF 2.0
mass % mass% mass% mass% cmolc/kg g/kg mg/kg mass %
1 4.4 30.7 26 15 59 41.7 11.7 3 85
2 5.0 2.0 87 10 3 4.2 1.9 1 23
3 5.2 2.8 88 10 2 6.3 1.5 <1 27
4 5.2 1.8 89 9 2 4.1 1.0 1 19
5 6.3 3.6 50 19 31 30.0 15.3 1 37
6 6.7 0.9 9 78 13 12.2 2.2 1 34
7 6.8 0.6 54 12 35 14.2 0.7 2 28
8 7.3 1.3 25 63 13 14.3 1.2 1 31
9 7.6 2.1 15 64 21 24.8 0.5 1 34
10 7.8 0.8 56 26 18 14.1 0.1 1 23

In addition, also a standard OECD artificial soil was included for invertebrate tests: pH 7.0, 70% sand, 20% kaolin clay and 10% spaghnum peat

Nominal and measured concentrations:

Nominal concentrations (mg/kg)
soil 1 : 0 (control), 32, 100, 320, 1000, 3200
soil 2 : 0 (control), 3.2, 10, 32, 100, 320, 1000, 3200
soils 3 to 10 and OECD soil: 0 (control), 10, 32, 100, 320, 1000, 3200
measured concentrations (mg/kg)
soil 1 : 4, 33, 107, 288, 993, 2722
soil 2 : 1, 3, 8, 26, 80, 239, 960, 2694
soil 3 : 3, 8, 26, 78, 242, 836, 2369
soil 4 : 0, 10, 30, 89, 277, 916, 2588
soil 5 : 2, 10, 26, 80, 255, 799, 3396
soil 6 : 1, 9, 29, 88, 284, 928, 2896
soil 7 : 2, 10, 25, 70, 242, 763, 2744
soil 8 : 1, 8, 27, 83, 214, 742, 2844
soil 9 : 1, 9, 27, 79, 291, 856, 2817
soil 10 : 1, 8, 25, 78, 226, 867, 2821
OECD soil : 1, 8, 28, 86, 279, 958, 2628

Nominal concentrations (mg/kg dw soil) for the aged soils at time 0
soil 4, 5 and 6 : 0 (control), 10, 32, 100, 316, 1000, 3200, 10000
Measured concentrations (mg/kg dw soil) for the aged soil at time 0
soil 4 : 1(control), 9, 27, 86, 269, 838, 2900, 10135
soil 5 : 6(control), 11, 28, 89, 290, 913, 3158, 11137
soil 6 : 6(control), 10, 34, 91, 268, 908, 3295, 9365
Measured concentration (mg/kg dw soil) for the aged soils at time 11-months
soil 4 : <1 (control), 2, 4, 13, 9, 10, 9, 78
soil 5 : 4 (control), 5, 13, 25, 25, 58, 2444, 5540
soil 6 : 3 (control), 6, 10, 12, 16, 694, 2789, 4485

Results and discussion

Bioconcentration factor

Bioconcentration factor 1

Type:

BSAF

Value:

0.35

Basis:

whole body d.w.

Calculation basis:

other: Mean BAF of exposure concentrations below the EC10 and omitting values for the controls

Remarks on result:

other: soil 1

Bioconcentration factor 2

Type:

BSAF

Value:

2.45

Basis:

whole body d.w.

Calculation basis:

other: Mean BAF of exposure concentrations below the EC10 and omitting values for the controls

Remarks on result:

other: soil 2

Bioconcentration factor 3

Type:

BSAF

Value:

1.73

Basis:

whole body d.w.

Calculation basis:

other: Mean BAF of exposure concentrations below the EC10 and omitting values for the controls

Remarks on result:

other: soil 3. (Mean BAF taken from earthworms exposed below 80 mg/kg , that is, just above the EC10)

Bioconcentration factor 4

Type:

BSAF

Value:

1.94

Basis:

whole body d.w.

Calculation basis:

other: Mean BAF of exposure concentrations below the EC10 and omitting values for the controls

Remarks on result:

other: soil 4

Bioconcentration factor 5

Type:

BSAF

Value:

0.52

Basis:

whole body d.w.

Calculation basis:

other: Mean BAF of exposure concentrations below the EC10 and omitting values for the controls

Remarks on result:

other: soil 5. BAF = 0.47 when omitting an obvious outlier at 80 mg Mo/kg exposure concentration

Bioconcentration factor 6

Type:

BSAF

Value:

1.03

Basis:

whole body d.w.

Calculation basis:

other: Mean BAF of exposure concentrations below the EC10 and omitting values for the controls

Remarks on result:

other: soil 6. (Mean BAF taken from earthworms exposed below 30 mg/kg, well above the EC10)

Bioconcentration factor 7

Type:

BSAF

Value:

0.51

Basis:

whole body d.w.

Calculation basis:

other: Mean BAF of exposure concentrations below the EC10 and omitting values for the controls

Remarks on result:

other: soil 7

Bioconcentration factor 8

Type:

BSAF

Value:

2.08

Basis:

whole body d.w.

Calculation basis:

other: Mean BAF of exposure concentrations below the EC10 and omitting values for the controls

Remarks on result:

other: soil 8. Mean BAF taken from earthworm exposed to concentrations up to the EC50

Bioconcentration factor 9

Type:

BSAF

Value:

1.88

Basis:

whole body d.w.

Calculation basis:

other: Mean BAF of exposure concentrations below the EC10 and omitting values for the controls

Remarks on result:

other: soil 9. (Mean BAF taken from earthworms exposed below 30 mg/kg, well above the EC10)

Bioconcentration factor 10

Type:

BSAF

Value:

3.44

Basis:

whole body d.w.

Calculation basis:

other: Mean BAF of exposure concentrations below the EC10 and omitting values for the controls

Remarks on result:

other: soil 10. (Mean BAF taken from earthworms exposed below 30 mg/kg, well above the EC10)

Bioconcentration factor 11

Type:

BSAF

Value:

0.44

Basis:

whole body d.w.

Calculation basis:

other: Mean BAF of exposure concentrations below the EC10 and omitting values for the controls

Remarks on result:

other: soil 11 (OECD soil)

Bioconcentration factor 12

Type:

BSAF

Value:

1.92

Basis:

whole body d.w.

Calculation basis:

other: Mean BAF of exposure concentrations below the EC10 and omitting values for the controls

Remarks on result:

other: 11-month aged Soil 4

Bioconcentration factor 13

Type:

BSAF

Value:

0.54

Basis:

whole body d.w.

Calculation basis:

other: Mean BAF of exposure concentrations below the EC10 and omitting values for the controls

Remarks on result:

other: 11-month aged soil 5

Bioconcentration factor 14

Type:

BSAF

Value:

0.84

Basis:

whole body d.w.

Calculation basis:

other: Mean BAF of exposure concentrations below the EC10 and omitting values for the controls

Remarks on result:

other: 11-month aged soil 6

Details on results:

Bioaccumulation data were obtained for all freshly spiked soils as well as for the 11-month aged soils. In soils 1,3, 4, 5, 8 and the 11-month aged soils, earthworm Mo concentrations increased linearly with increasing soil concentrations. in soil 2, earthworm concentrations seemed to level off and reach a maximum at higher exposure levels. In soils 6, 7,9, 10 and 11, earthworm concentrations peaked at an intermediate exposure level. But also in these soils, earthworm Mo concentrations increased linearly with increasing soil concentrations in the non-toxic range. BSAF values below 1 were found for soils 1, 5, 7 and 11, while BSAF was around 1 for soil 6. BSAF was highest in soils 10, 2 and 8 with values >2.0 (but below 3.5).

Reported statistics:

BSAF for the accumulation of Mo in earthworms were calculated by dividing concentrations in the surviving animals by measured total concentrations in the test soils. BSAFs were calculated as the mean of the BSAFs for all concentrations at which earthworms survived. BSAF values for metals may show deviating values at higher exposure levels due to toxicity. BSAFs may also deviate for control soils compared to spiked soils. For this reason, also BSAFs were calculated omitting higher concentration levels (>EC10)and controls.
Soil properties most relevant for bioaccumulation of Mo in the test organisms were identified by correlating BSAF with soil properties using Pearson correlation analysis and simple and multiple stepwise regression analysis. All analysis were run in the software package SPSS 15.0 for Windows. Simple regression revealed ammonium oxalate extractable iron (Feox) as the most relevant parameter: BSAF = 1.809 – 1.208 log Feox (R2 adjusted = 0.517; p=0.012; n=9) Stepwise regression extended this equation with ammonium oxalate extractable P (Pox) to read: BSAF = 2.326 – 1.933 log Feox + 1.199 log Pox (R2 adjusted = 0.778; p=0.002; n=9). Together, these parameters explained 78% of the variation in the BSAF values.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Molybdenum concentrations in earthworms showed a dose-related increase with increasing soils concentrations in most soils. In some soils earthworm concentrations peaked at lower soil concentrations, probably as a consequence of toxicity. But also in these soils Mo concentrations in the earthworms increased linearly with soil concentrations in the non-toxic range, e.g. below the EC10. These data suggest that although being an essential element, Mo is not regulated by the earthworms to constant body concentrations like e.g. Zn. However, BSAF values obtained at soil concentrations in the non-toxic range are all low, with BSAFs between 0.35 to 3.44. In addition, remarkably good agreement was seen between BSAF values obtained for the freshly spiked and the 11-month aged soils, suggesting that biavailability was not affected by ageing of the soils. This data can be used for the determination of BSAFs for the soil to worm trophic transfer.