The product from the burning of a combination of carbonaceous materials.

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP compliant, non-guideline experimental study. Study published in scientific, peer reviewed journal.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

Annelid worms were exposed to soil simulating coniferous forest floor with humus and litter layers, and a seedling of silver birch with or without wood ash amendments in laboratory using microcosms.

GLP compliance:

no

Details on sampling:

First sampling 15 weeks after the start of the experiment, second sampling week 17, third sampling week 27, fourth sampling week 54. At each sampling five replicates per treatment were taken. Enchytraeids were extracted using a wet funnel method, and 55 individuals, corresponding to 10 166 individuals per m2, were introduced to 20 ash-free and 20 ash-treated microcosms.

Details on preparation and application of test substrate:

Wood ash which was carefully mixed into the soil and litter, and the other part was left as ash free controls. Each ash treated microcosm received 2,8 g wood ash corresponding to 5000 kg wood ash per hectare. Microarthropods were later reintroduced into the systems.

Test organisms (species):

Cognettia sphagnetorum

Animal group:

annelids

Study type:

laboratory study

Substrate type:

natural soil

Total exposure duration:

54 wk

Test temperature:

Incubation conditions were set up to simulate summer (+17°C), autumn (day +7°C, night + 5°C), winter (+2,5-+4°C) and spring (day +7°C, night +5°C).

Nominal and measured concentrations:

Ca 350 mg/kg; Mg 17mg/kg;K 26 mg/kg; P 9 mg/kg; Mn 10 mg/kg; Zn 2,5 mg/kg; Cd 15 mg/kg; Al 12 mg/kg; Cu 72 mg/kg; Fe 9 mg/kg

Reference substance (positive control):

no

Remarks on result:

other: Effect concentration was not determined

Details on results:

C. sphagnetorum was very sensitive to application of wood ash; their numbers and biomass were substantially lower in the ash treated microcosms.
see illustration: Biomass of Cognettia sphagnetorum before (the baseline sampling) and immediately after the drought (the resistance sampling), and 10 and 37 weeks after the drought (the 1st and 2nd resilience samplings) in the ash-free and ash-treated soil

C. sphagnetorum was very sensitive to application of wood ash; their numbers and biomass were substantially lower in the ash treated microcosms (see illustration). However, effect concentrations were not determined in the study.

Validity criteria fulfilled:

not applicable

Conclusions:

C. sphagnetorum was very sensitive to application of wood ash; their numbers and biomass were substantially lower in the ash treated microcosms.

Executive summary:

A non-GLP, non-guideline laboratory microcosm experiment was established to study chronic effects of wood ash amendments on annelid Cognettia shpagnetorum. Simulated coniferous forest floor with humus and litter layers, and a seedling of silver birch (Betula pendula) were established with or without wood ash amendment. C. sphagnetorum was very sensitive to application of wood ash; their numbers and biomass were substantially lower in the ash treated microcosms.C. sphagnetorum is a keystone species in acid forest soils. Since ash application decreases soil acidity, the activity of C. sphagnetorum in the detrital food web is affected.

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP compliant, non-guideline experimental investigation. Study published in scientific, peer reviewed journal.

Qualifier:

no guideline available

Principles of method if other than guideline:

Acidic coniferous forest soil was treated with wood ash at 5000 kg ha−1 or left as ash-free control. The microcosms were destructively sampled 26 and 51 weeks after initiation of the experiment. Enchytraeid biomass was measured at the end of the experiment.

GLP compliance:

no

Details on sampling:

The microcosms were destructively sampled twice during the experiment. The number of replicates at the first sampling was three and at the second sampling five.

Details on preparation and application of test substrate:

The humus layer [pH in deionised water 4.2 (ISO 10390), water content 65% of fresh mass (ISO 11465)] used in the study was collected from a pine forest near the city of Jyväskylä, central Finland, sieved through a 5.6-mm sieve and autoclaved. The acidity of the humus increased during autoclaving and therefore 8 mg CaCO3 to 10 g fresh mass humus was added. After CaCO3 addition the soil pH was 4.5. Litter materials, consisting mainly of pine needles, were collected from the same forest as the humus and were also autoclaved.

The microcosms were sterilised with Virkon® S-solution (Antec Int. Ltd.) before the forest floor was constructed in the microcosms by spreading humus (thickness of the layer was 3 cm, dry mass 44.2 g) on the bottom of each microcosm and adding a litter layer about 0.5 cm thick (4.2 g d.m.) on the humus. In half of the microcosms 2.8 g d.m. of wood ash corresponding to 5000 kg d.m. of wood ash per hectare was mixed into the soil (2 g in the humus and 0.8 g in the litter). After wood ash application the pH of the humus was 6.2 and that of the litter 7.8.

Test organisms (species):

Cognettia sphagnetorum

Animal group:

annelids

Details on test organisms:

Enchytraeids were extracted from raw humus soil using a wet funnel method. In order to reduce the amount of microbes possibly being introduced to the microcosms with enchytraeid worms, the worms were stored for a week in sterilised water, which was changed twice during the storing.

Study type:

laboratory study

Substrate type:

artificial soil

Total exposure duration:

51 wk

Details on test conditions:

Enchytraeids were introduced into the microcosms. Half of the ash-free and half of the ash-treated microcosms received 30 enchytraeid individuals, corresponding to 6000 individuals m−2. The microcosms were placed in randomised order in a climate chamber and incubated for two growing periods (+17 °C, daily cycle of 16 h light, illumination 600 μE m−2 s−1) for the pine, with artificially created autumn (duration 2 weeks; temperature during the day +7 °C and during the night +5 °C, daily cycle of 11 h light, illumination 250 μE m−2 s−1) and winter (5 weeks, +2.5 °C, daily cycle of 5 h light, illumination 150 μE m−2 s−1). The two growing periods took place between weeks 0 and 26 and 34 and 51, with the autumn and winter taking place between weeks 27 and 33. The amount of water that evaporated during the experiment was evaluated by weighting the microcosms and the water lost was replaced with sterilised water.

Nominal and measured concentrations:

The wood ash contained 350 mg kg−1 Ca, 17 mg kg−1 Mg, 26 mg kg−1 K, 9 mg kg−1 P, 10 mg kg−1 Mn, 2.5 mg kg−1 Zn, 15 mg kg−1 Cd, 12 mg kg−1 Al, 72 mg kg−1 Cu, and 9 mg kg−1 Fe.

Duration:

51 wk

Remarks on result:

not measured/tested

Remarks:

Enchytraeid biomass was the measured endpoint in the study. No LC-, EC-, NOEC or LOEC values were determined.

Details on results:

Enchytraeid biomass was lower in the wood ash-treated soil than in the ash-free soil at both samplings (the first sampling: F = 17.09, P = 0.004 and the second sampling: F = 15.16, P = 0.002).

Reported statistics and error estimates:

- Enchytraeid biomass was lower in the wood ashtreated soil than in the ash-free soil at both samplings (the first sampling: F = 17.09, P = 0.004 and the second sampling: F = 15.16, P = 0.002).
- Soil nematodes were mainly unresponsive to the treatments; the only statistically significant effect was the reduction in fungal feeding nematodes in the wood ash-treated soils at the first sampling
-Wood ash had a clear negative effect on pine seedling growth at the second sampling when only if enchytraeids (F = 29.11, P < 0.001) or EM-fungi (F = 12.75, P < 0.01) were alone in the microcosms.
- Enchytraeids stimulated plant growth in the ashtreated microcosms (F = 4.68, P < 0.05) and in the ashfree microcosms without EM-fungi (F = 23.71, P < 0.001).
- EM-fungi enhanced pine growth in the absence of enchytraeids (F = 7.86, P < 0.01), and in the soils treated with wood ash (F = 4.89, P < 0.05).
- EM- fungi tended to reduce pine growth in the ash-free soils with enchytraeids by 28% (F = 4.36, limit for P = 0.05 is F = 4.47).
- Enchytraeids increased root length in the wood ash-treated soils (F = 9.5, P < 0.01) and in the absence of EM-fungi (F = 15.95, P < 0.001).
- EM-fungi had a positive effect on root length in the microcosms without enchytraeids (F = 7.60, P < 0.05) and a negative effect in the microcosms with ash-free soil and enchytraeids (F = 4.73, P < 0.05).
- At the first sampling, application of wood ash to the soil decreased the amount of water extractable NH4 +-N in the presence of enchytraeids (F = 17.19, P < 0.001).
- Wood ash treatment decreased the amount of water extractable NO3-N in the presence of enchytraeids (F = 10.46, P < 0.01). Enchytraeids stimulated leaching of NO3-N in the absence of EM- fungi (F = 6.13, P < 0.05) and decreased it in the presence of EM-fungi (F = 4.93, P < 0.05). EM-fungi
increased the amount of water extractable NO3-N in the absence of enchytraeids (F = 9.31, P < 0.01).

Wood ash had a clear negative impact on enchytraeid populations.

Validity criteria fulfilled:

not specified

Conclusions:

Wood ash had a clear negative impact on enchytraeid populations.

Executive summary:

A non-GLP, non-guideline experiment was conducted in laboratory microcosms to study the chronic effects of wood ash amendments enchytraeid Cognettia shpagnetorum.Simulated boreal coniferous forest floor with humus and litter layers and a seedling of Scots pine (Pinus sylvestris) were prepared, and wood ash corresponding to 5000 kg d.m. of wood ash per hectare was added to half of the microcosms. Wood ash had a clear negative impact on enchytraeid population since there was a decrease in biomass.

Reference 3

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP compliant, non guideline experimental study. Study published in a scientific, peer reviewed journal.

Qualifier:

no guideline available

Principles of method if other than guideline:

A laboratory microcosm experiment with two main factors: wood ash (without ash, ash spread on the soil surface and ash mixed into the soil), and C. sphagnetorum (with/without) was established using a simulated coniferous forest floor with humus and litter layers, and a seedling of Scots pine (Pinus sylvestris).
The experiment was conducted in 48+5 transparent plastic bottles (diameter 8 cm, height of 28 cm), which were sealed with cotton wads to allow free gas exchange in the microcosms.

GLP compliance:

no

Test organisms (species):

Cognettia sphagnetorum

Animal group:

annelids

Study type:

laboratory study

Substrate type:

artificial soil

Total exposure duration:

51 wk

Duration:

51 wk

Remarks on result:

not measured/tested

Remarks:

Biomass of enchytraeids was the measured endpoint in the study. No LC-, EC-, NOEC or LOEC values were determined.

Reported statistics and error estimates:

- Biomass of enchytraeids was reduced by the wood ash (F = 8.78, P = 0.003), but only when it was mixed into the soil (control versus ash mixed P = 0.001).
- In the microcosms where ash was spread on the soil surface the enchytraeid biomass was between the other two treatments, but it differed significantly only from the ash mixed into the soil treatment (P = 0.01).
- Enchytraeid biomass decreased towards the end of the experiment as their biomass was lower at the second than at the first sampling (F = 28.67, P <0.001).

Biomass of enchytraeids was reduced by the wood ash, but only when it was mixed into the soil. In the microcosms where ash was spread on the soil surface the enchytraeid biomass was between the other two treatments, but it differed significantly only from the ash mixed into the soil treatment. Enchytraeid biomass decreased towards the end of the experiment as their biomass was lower at the second than at the first sampling.

Conclusions:

Cognettia sphagnetorum is sensitive to application of wood ash. However, enchytraeids can maintain nutrient cycling and thus ensure plant growth under disturbed conditions (such as increase in pH due to ash application) even when their population density is low.

Executive summary:

A non GLP, non Guideline laboratory microcosm experiment was established to study whether the role of Cognettia sphagnetorum (Enchytraeidae) in affecting Scots pine (Pinus sylvestris) seedling growth is influenced by wood ash-amendment, i.e.,

neutralisation of the raw humus soil.Wood ash when mixed into the soil, reduced the biomass of C. sphagnetorum. The presence of enchytraeids in the ash-treated soil compensated the ashinduced negative effects on the pine performance. Thus, C. sphagnetorum can be important in ensuring nutrient cycling and plant growth in situations when an ecosystem encounters disturbances

Reference 4

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP compliant, non-guideline experimental investigation. Study published in scientific, peer reviewed journal.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Adult earthworm Lampito mauritii were exposed to different proportions of fly ash in soil for 30 d and the concentrations of metallothionein in earthworm were assessed.

GLP compliance:

no

Remarks:

A WOE study published in a peer reviewed scientific journal

Details on sampling:

For each assay four worms from four different replicates of each proportion of fly ash (specified for a particular day) were selected randomly at the end of the experimental exposure. The earthworms were collected carefully at 0 (zero) and 30 d of fly ash exposure and kept overnight on a moist filter paper without food for evacuating their gut contents. These animals were weighed, sacrificed by freezing.

Details on preparation and application of test substrate:

Two types of experimental beds containing 300 gm of culture materials were prepared as follows: (i) fly ash amended bed (soil + FYM + fly ash + earthworm) (ii) control bed (soil + FYM + earthworm). To obtain fly ash amended beds fly ash was added in each bed so as to make the percentage in the experimental beds as 5%, 10%, 15%, 25%, 40% and 50%. The balance amount in each bed was maintained with a mixture of fine sieved air dried soil (2 mm) and FYM in the ratio of 9:1. The soil was mixed thoroughly to ensure a homogenous mixture. The moisture content was adjusted to 30% of the final weight in all experimental beds. The average height of each bed was 10 cm and initial pH of the soil was 6.8 ± 0.05.The temperature was maintained at 28 ± 2 C throughout the study period. The experimental pots were left for 15 d undisturbed prior to experimentation for softening of wastes or thermo stabilization.

Test organisms (species):

other: Lampito mauritii

Animal group:

annelids

Details on test organisms:

The earthworm species, L. mauritii is believed to be indigenous to Indo-Pacific region. L. mauritii is well equipped to metabolize electrophilic xenobiotics and thus gets easily adapted to metal (Pb and Zn) stressed environment. In this species of earthworm, reproduction is sexual and biparental andthe average number of cocoons produced by each adult in a year is 14.25 with 4 weeks of incubation period. High rate of cocoon production, short development time with high hatching success, as well as continuous breeding strategies in L. mauritii indicate their possible usefulness in vermiculture. This detrivorous anecic species has the ability to survive in a wide range of soil pH (5.8–7.2) and can also adjust well in the laboratory.
The earthworms (L. mauritii) were collected from the meadow, which had no history of input of either heavy metals or agrochemicals. They were carefully brought to the laboratory along with the moist soil and acclimatized for 1 month under laboratory conditions in polyethylene buckets containing soil and farmyard manure mixture (FYM) in the ratio of 9:1. The adult earthworms were removed from the culture pots 24 h prior to their use, rinsed in water and kept on moist filter paper in the dark at 28 ± 2 C as a routine procedure to allow voiding of gut contents.

Study type:

laboratory study

Substrate type:

natural soil

Total exposure duration:

30 d

Remarks:

.

Test temperature:

28 ± 2 oC

pH:

6.8 ± 0.05 (initial)

Moisture:

30 %

Details on test conditions:

Eighteen adult (well clitellate) gut evacuated earthworms were inoculated in each experimental bed and control bed. Culture pots were covered with nylon net to prevent escape of earthworms.

Nominal and measured concentrations:

Metal Content (%)
in soil: Si 81.41, Fe 2.158, Ca 0.958, K 0.958, Mg 0.115, Mn 0.0684, Pb 0.00104, Zn 0.0146, Na 0.0864, Ni 0.00038, Cd 0.00112, Cr 0.022, Cu 0.00492
in FYM Si 31.8, Fe 2.417, Ca 1.365, K 1.365, Mg 0.569, Mn 0.091, Pb 0.013, Zn 0.019, Na 0.1098, Cr 0.056,
in fly ash Si 52.23, Fe 6.325 Ca 1.463, K 0.63, Mg 1.25, Pb 0.027, As 0.0025, Zn 0.046, Na 3.06, Ni 0.2624, Cd 0.0026, Cr 0.039, Cu 0.009

Duration:

30 d

Remarks on result:

not measured/tested

Remarks:

Endpoints used in the study included mortality and body weight. No LC-, EC-, NOEC or LOEC values were determined.

Details on results:

There was no significant alteration in body weight of the earthworm in fly ash amended beds with respect to the control ones for the specific period of exposure. No mortality was also observed throughout the experiment. Prior to exposure (0 d), Zn was found to be 28 mg/kg and Pb, Cd, As, Cr, Ni and Cu were below detection limit of the atomic absorption spectrometer in the wet body mass of earthworm. However, no significant change was observed in the analyzed metal concentrations of earthworm tissue after 30 d of fly ash exposure. However, the MT content of earthworm from the fly ash amended beds remained significantly higher than that of the control at all days of exposure. After 5 and 10 d of post-exposure to 5% fly ash, 27.32% and 45.17% induction in MT level were recorded respectively. After 5 d of exposure to 10%, 15%, 25%, 40% and 50% of fly ash, the MT concentration demonstrated 41.37%, 41.13%, 37.46%, 39.57% and 44.66% induction respectively in the earthworms. Ten days of 10–50% fly ash amendment showed 51.86–59.06% MT induction in L. mauritii. After 15 d, the highest rate of MT induction was recorded at 15– 40% of fly ash amended soil exposure. However, at all proportions of fly ash amendment, the highest MT induction was recorded after 10 d of exposure and the minimum was recorded after 30 d of exposure.

Reported statistics and error estimates:

The experimental values of tissue MT concentrations in the earthworms were expressed as mean ± standard error (SE) of three replicates, each comprising four worms. The test of significance was performed by one-way analysis of variance followed by Duncan Multiple Range test considering P < 0.05.

No significant change was observed in the analyzed metal concentrations of earthworm tissue after 30 d of fly ash exposure. However, the MT content of earthworm from the fly ash amended beds remained significantly higher than that of the control at all days of exposure. At all proportions of fly ash amendment, the highest MT induction was recorded after 10 d of exposure (51.86–59.06%) and the minimum was recorded after 30 d of exposure.

Conclusions:

The results revealed that up to 50% of fly ash amendment does not apparently harm the earthworm in respect of their survival and groth. A significant increase in tissue metallothionein level was recorded in L. mauritii exposed to fly ash amended soil without tissue metal accumulation indicating that metallothionein is involved in scavening of free radicals and reactive oxygen species metabolites.

Executive summary:

Adult earthworm Lampito mauritii were exposed to different proportions of fly ash in soil in a 30 -d non-GLP compliant and non-guideline experimental study. Test material was prepared from soil and farmyard manure mixture (FYM) in the ratio of 9:1. The soil for beds was taken from the site of collectionof earthworms, which has no history of input of heavy metals and agrochemicals. To obtain fly ash amended beds fly ash was added in each bed so as to make the percentage in the experimental beds as 5%, 10%, 15%, 25%, 40% and 50%. The concentrations of metals and metallothionein (MT) in earthworm were assessed after the experiment.

There was no significant alteration in body weight of the earthworm in fly ash amended beds with respect to the control ones for the specific period of exposure. No mortality was also observed throughout the experiment. No significant change was observed in the analyzed metal concentrations of earthworm tissue after 30 d of fly ash exposure. However, the MT content of earthworm from the fly ash amended beds remained significantly higher than that of the control at all days of exposure. At all proportions of fly ash amendment, the highest MT induction was recorded after 10 d of exposure (51.86–59.06%) and the minimum was recorded after 30 d of exposure.

Description of key information

Terrestrial toxicity is estimated based on four scientific publications from literature. In three of them chronic effects of ash amendments on annelid Cognettua shpagnetorum were studied in microcosms. The studied effects included direct effects (survival and growth of annelids ), indirect effects (impacts from presense of annelids on plant growth through nutrient cycling) and population level effects. In the fourth investigation, effects of ash to earthworm Lampito mauritii was studied in a laboratory. 
Enchytraeid worm (C. sphagnetorum) was very sensitive to ash; their number and biomass decresed substantitally in the ash treated microcosms. Instead, survival and growth of earthworm (L. mauritii) was not affected by up to 50% of fly ash amendment.The results indicated that metallothionein acts as scavengers of free radicals and reactive oxygen species metabolites.

Additional information

The disappearance of key functional species (like C. sphagnetorum in acid forest soils) or groups does not, however, necessarily result in perpetual losses of fundamentally important ecosystem-level functions provided that a new, hitherto non-existing functional species can invade the disturbed area. Moreover, many soil organisms are of omnivorous nature and share a a high degree of functional redundancy.