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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well documented studies, according to international guidelines
Qualifier:
according to
Guideline:
OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)
Version / remarks:
with 24h respiration as an endpoint
GLP compliance:
yes
Remarks:
The study was carried out in compliance with the principles of good laboratory practice.
Analytical monitoring:
yes
Vehicle:
no
Details on preparation and application of test substrate:
Spiking: Air-dried and sieved soil samples were spiked with PbCl2 (2 g Pb/L) to 7 concentrations (control plus 6 treatments). Additional deionized water was added together with the spike solution to adjust the soil moisture content to 75% of pF 2.0. In case the desired Pb dose was not reached with the spike solutions, PbCl2 was mixed in the soil as crushed powder. All soils were thoroughly mixed after amendments using laboratory spoons. Moisture content in control soils was raised to 75% of pF 2.0 with deionized water only. Spiked soils were air-dried and sieved again and stored pending toxicity testing.
Leaching: A portion of the spiked soils was not dried but dark-incubated at 20°C for a week where after these were leached and pH corrected. Soils were leached with “artificial rain water” (ARW, 5*10-4 M CaCl2, 5*10-4 M Ca(NO3)2, 5*10-4 M MgCl2, 10-4 M Na2SO4, 10-4 M KCl, pH 5.9) in order to avoid soil peptisation and to replace nutrients that were leached. The leaching volume was about 2 pore volumes. After leaching the soils were air dried and pH values were adjusted with CaO to obtain no larger pH differences than 0.2 per soil.
Ageing: In 2005 the same spiking and leaching procedure was followed for soil 3 soils. However, these soils were spiked with Pb(NO3)2 to 7 concentrations (control plus 6 treatments). The new spiking (2010) was performed with PbCl2 instead of Pb(NO3)2 because of the hormesis obtained for plant growth tests after NO3- amendments. Five kg (dry weight) of each treatment was stored in perforated flower pots, which were incubated outside in sand. In this way, the soils were left to age for 5 years. No pH correction was done before incubation outside, but pH was corrected after ageing.

All spiked, leached, and aged soils were dry stored prior to toxicity testing. Ageing reactions in the air dried soils are neglected during the period between spiking and toxicity testing. For all dose-response studies, soils are wetted to 75 % of field capacity 2 weeks in advance and are thoroughly mixed after that period before testing.
Test organisms (inoculum):
soil
Total exposure duration:
24 h
Test temperature:
20°C
Details on test conditions:
Prior to the respiration test, the air-dried Pb(NO3)2/PbCl2 spiked soils were pre-incubated at a moisture content equivalent to 75 % of that at pF 2.0 for 14 days at 20°C. After this incubation period 5 g subsamples of each Pb concentration (of each soil) were placed in 20 ml plastic pots. Soils were then amended with 0.125 mL of 14C labeled glucose solution (40 mg/mL glucose, specific activity = 2.3 KBq/mg glucose-C) and mixed thoroughly. The plastic pots with soil were placed in airtight Schott bottles of 100 ml with 5 ml of NaOH 1 N at the bottom. These Schott bottles were incubated at 20°C in dark for 24 hours. Upon completion of the incubation period NaOH traps were removed and 1.0 mL subsamples were taken and added to 10 mL scintillation cocktail (Ultima Gold), with the solutions then shaken and activity determined by beta counting. The percentage added glucose-C which was respired, was determined, and Pb dose response curves were derived based on soil (mg Pb/kg) concentrations.

- 7 uncontaminated soils from various European regions were sampled. Soils were collected with a metal spade from the plough layer, or from the surface horizons for grassland soils. Soils were dried, sieved and stored in darkness.

Soil properties:
pH organic C org. matter clay background Pb eCEC
% % % mg Pb/kg cmolc/kg
Natural soil, Barcelona, leached 7.4 1.4 2.4 16 135 14.7
Natural soil, Woburn, leached 6.5 3.1 5.3 30 46 27.1
Natural soil, Ter Munck, leached 6.7 1.0 1.7 12 22 8.7
Natural soil, De Meern, leached 5.3 5.0 8.6 60 52 42.0
Natural soil, Borris, leached 5.7 1.5 2.6 3 15 4.2
Natural soil, Hygum, leached 5.2 2.1 3.6 13 18 7.6
Natural soil, Kasterlee, leached 4.9 2.7 4.7 2 28 4.2
Natural soil, Barcelona, freshly spiked 7.4 1.2 2.1 16 137 14.3
Natural soil, Woburn, freshly spiked 6.1 4.3 7.4 30 52 26.5
Natural soil, Ter Munck, freshly spiked 6.2 1.0 1.7 12 21 8.4
Natural soil, Kasterlee, freshly spiked 4.7 2.3 4.0 2 24 4.0
Natural soil, Barcelona, 5 years aged 7.0 7.3 12.6 18 126 14.4
Natural soil, Woburn, 5 years aged 6.7 3.3 5.7 21 102 22.3
Natural soil, Ter Munck, 5 years aged 6.6 1.4 2.4 9 29 8.2
Nominal and measured concentrations:
total measured concentrations: - Barcelona freshly spiked:137, 456, 763, 1200, 1970, 5580 and 8720 mg Pb/kg - Barcelona leached: 135, 435, 580, 933, 1960, 4410 and 7190 mg Pb/kg - Barcelona aged: 126, 424, 645, 1070, 1900, 4020 and 7020 mg Pb/kg - Woburn freshly spiked: 52, 306, 579, 891, 2050, 2960 and 6450 mg Pb/kg - Woburn leached: 46, 266, 479, 753, 1270, 3120 and 5020 mg Pb/kg - Woburn aged: 102, 276, 301, 605, 1670, 3300 and 5620 mg Pb/kg - Ter Munck freshly spiked: 21, 226, 449, 777, 1870, 4560 and 6620 mg Pb/kg - Ter Munck, leached: 22, 250, 472, 793, 1950, 3090 and 7130 mg Pb/kg - Ter Munck, aged: 29, 518, 673, 1010, 1700, 4279 and 6410 mg Pb/kg - De Meern leached: 52, 139, 291, 841, 2726, 9320 and 23300 mg Pb/kg - Borris leached: 15, 34, 73, 169, 444, 1030 and 3380 mg Pb/kg - Hygum leached: 18, 47, 93, 259, 628, 2450 and 6184 mg Pb/kg - Kasterlee freshly spiked: 24, 36, 59, 131, 316, 762 and 2080 mg Pb/kg - Kasterlee leached: 28, 34, 47, 114, 374, 844 and 1940 mg Pb/kg nominal added doses: - Barcelona, Woburn and Ter Munck: 0, 250, 500, 1000, 2000, 4000 and 8000 mg Pb/kg - De Meern: 0, 99, 311, 994, 3105, 9936 and 31050 mg Pb/kg - Borris: 0, 17, 52, 166, 518, 1656 and 5175 mg Pb/kg - Hygum: 0, 23, 72, 232, 725, 2318 and 7245 mg Pb/kg - Kasterlee: 0, 10, 31, 99, 311, 994 and 3105 mg Pb/kg
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
573 mg/kg soil dw
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Pb
Basis for effect:
respiration rate
Remarks:
glucose induced respiration
Remarks on result:
other: Barcelona soil, 5 years aged after spiking
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
2 380 mg/kg soil dw
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Pb
Basis for effect:
respiration rate
Remarks:
glucose induced respiration
Remarks on result:
other: Woburn soil, 5 years aged after spiking
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
1 320 mg/kg soil dw
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Pb
Basis for effect:
respiration rate
Remarks:
glucose induced respiration
Remarks on result:
other: Ter Munck soil, 5 years aged after spiking
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
2 300 mg/kg soil dw
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Pb
Basis for effect:
respiration rate
Remarks:
glucose induced respiration
Remarks on result:
other: Barcelona soil, leached and pH corrected after spiking
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
1 120 mg/kg soil dw
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Pb
Basis for effect:
respiration rate
Remarks:
glucose induced respiration
Remarks on result:
other: Woburn soil, leached and pH corrected after spiking
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
385 mg/kg soil dw
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Pb
Basis for effect:
respiration rate
Remarks:
glucose induced respiration
Remarks on result:
other: Ter Munck soil, leached and pH corrected after spiking
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
>= 23 300 mg/kg soil dw
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Pb
Basis for effect:
respiration rate
Remarks:
glucose induced respiration
Remarks on result:
other: De Meern soil, leached and pH corrected after spiking
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
179 mg/kg soil dw
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Pb
Basis for effect:
respiration rate
Remarks:
glucose induced respiration
Remarks on result:
other: Borris soil, leached and pH corrected after spiking
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
5 300 mg/kg soil dw
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Pb
Basis for effect:
respiration rate
Remarks:
glucose induced
Remarks on result:
other: Hygum soil, leached and pH corrected after spiking
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 940 mg/kg soil dw
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Pb
Basis for effect:
respiration rate
Remarks:
glucose induced respiration
Remarks on result:
other: Kasterlee soil, leached and pH corrected after spiking
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
6 090 mg/kg soil dw
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Pb
Basis for effect:
respiration rate
Remarks:
glucose induced respiration
Remarks on result:
other: Barcelona soil, no leaching or ageing of soil after spiking with Pb salt
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
306 mg/kg soil dw
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Pb
Basis for effect:
respiration rate
Remarks:
glucose induced respiration
Remarks on result:
other: Woburn soil, no leaching or ageing of soil after spiking with Pb salt
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
168 mg/kg soil dw
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Pb
Basis for effect:
respiration rate
Remarks:
glucose induced respiration
Remarks on result:
other: Ter Munck soil, no leaching or ageing of soil after spiking with Pb salt
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
>= 2 080 mg/kg soil dw
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Pb
Basis for effect:
respiration rate
Remarks:
glucose induced respiration
Remarks on result:
other: Kasterlee soil, no leaching or ageing of soil after spiking with Pb salt
Reported statistics and error estimates:
Dose-response curves were analyzed with measured added (i.e. corrected for background) concentrations and fitted by the log-logistic model using the newton optimization method. The ECx values, expressed as total Pb, are calculated from the sum of background Pb concentration of a soil and corresponding EDx (expressed as added Pb) fitted by the dose-response curves. Significant effects were determined by ANOVA (Dunnett's test) in order to determine the NOEC and LOEC values.
Validity criteria fulfilled:
yes
Conclusions:
Reliable study. The following EC10 values were selected for PNEC derivation: 573, 2380 and 1320 mg Pb/kg for aged Barcelona, Woburn and Ter Munck soils, respectively and 179 and 5300 mg Pb/kg for leached Borris and Hygum soils, respectively. Other toxicity values are presented as supporting information.

Description of key information

The chosen value is based on data for lead. The study was conducted according to ISO 14238.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:
7 020 mg/kg soil dw

Additional information